Method and apparatus for processing adipose tissue

ABSTRACT

A portable apparatus for collection and processing of human biological material, such as containing adipose extracted during a lipoplasty procedure, is useful for multi-step processing to prepare a concentrated product (e.g., stromal vascular fraction) or a fat graft composition. The apparatus has a container with a containment volume with a tissue retention volume and a filtrate volume separated by a filter and with a tapered portion to a collection volume for collecting concentrate product. Inlet and suction ports provide access to the tissue retention volume and filtrate volume, respectively, and an extraction port provides versatile access for removal of target processed concentrate material or fat graft material, which access may be via a lumen through a rotatable mixer shaft. Access ports may be configured for access only from above the container. A method of processing adipose tissue to concentrate leuko stromal vascular cells includes multi-step processing using a portable container.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation-in-part of U.S. patentapplication Ser. No. 13/808,550, which is a national stage ofInternational Patent Application No. PCT/US2011/043451, filed Jul. 8,2011, designating the United States of America, and which claimspriority to and thus the benefit of an earlier filing date from U.S.Provisional Patent Application No. 61/363,150, filed Jul. 9, 2010, theentire contents of each of which are hereby incorporated herein byreference. This application claims a benefit to U.S. Provisional PatentApplication No. 61/585,566, filed Jan. 11, 2012, the entire contents ofwhich are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods for processing human biologicalmaterial comprising adipose tissue, for example to prepare a concentratewith leuko stromal vascular cells, which include stem cells, andapparatus for processing human biological material, which may or may notinclude adipose tissue.

BACKGROUND OF THE INVENTION

Adipose tissue is recognized as a promising source of stem cells with atleast multi-potent differentiation potential. Lipoasperate obtainedduring a lipoplasty procedure, such as lipo surgery, may be processed toprepare a so-called stromal vascular fraction (SVF) that is rich inleuko stromal vascular cells, which include stem cells. Processing toprepare SVF may include washing lipoasperate with saline solution,followed by enzymatic digestion of washed tissue using collagenase, andcentrifuging digested material to prepare SVF in the form of acentrifuged pellet. Such collection and processing of tissue involvesseveral steps with transfer of contents between different processcontainers for different tissue collection and processing steps, whichis cumbersome and provides significant opportunities for error orcontamination.

Some attempts have been made to design portable containers in whichlipoasperate may be collected and then processed within the container todigest tissue and prepare a concentrate enriched in leuko stromalvascular cells. Potential benefits of using such portable containersinclude a reduced need to transfer material between containers toperform different process steps and a reduction in the need formultiple, specially-designed processing containers. However, suchmulti-step processing in portable containers faces significant equipmentand process design and operating limitations, especially when attemptingto process relatively large volumes of adipose tissue at one time.Desired leuko vascular cells, including stem cells, are sensitive toprocessing conditions and viability of recovered cells may suffersignificantly if processing is not adequately controlled. Also, recoveryof cells from the container is of critical importance. Significantpotential exists for loss of valuable cells to recovery from thecontainer, such as by cells adhering to internal equipment and surfaceswithin the container. One problem with multi-step processing in a singleportable container is that the container design and processingoperations must accommodate the different requirements of each of thedifferent process steps to be performed in the single container, andwith severe volume constraints in relation to a practical size for sucha portable container. In contrast, processing systems that involvetransfer of contents between multiple different containers forperformance of different process steps benefit from an ability tooptimize equipment and process design for each process container that isdedicated to performance of a single step of an overall process.Therefore, multi-container processing has significant advantages interms of step-by-step equipment and process optimization. Moreover, amulti-container design is better suited for automation, for example withautomated transfer of processed material through conduits betweendifferent process containers or with automated control of processparameters for uniformity and process control. Multi-containerprocessing remains the predominant processing technique at this time forprocessing adipose tissue to prepare stromal vascular fractionconcentrates.

SUMMARY OF THE INVENTION

The invention involves a portable apparatus adapted for multi-stepprocessing of human biological material. The human biological materialmay or may not comprise adipose tissue. The invention also involves amethod for multi-step processing of adipose tissue in a portablecontainer to prepare a concentrate product rich in leuko stromalvascular cells, or stromal vascular fraction cells.

A first aspect of the invention is provided by an apparatus that isadapted to serve both for collection of human biological material (e.g.,tissue, biological fluids) and for convenient multi-step post-collectionprocessing of collected material. Such human biological material andseparated or processed portions thereof may be referred to herein simplyas “tissue”, for convenience and brevity. For example, the term tissuemay be used herein to refer to in-tact tissue, disrupted tissue, tissuefragments and biological fluids associated with or separate from tissue.The apparatus is orientable in a collection orientation (also referredto as an access orientation) for collection of human biologicalmaterial, or tissue, for example which may comprise adipose tissuecollected during a lipoplasty procedure. The collection orientation isalso referred to herein as the access orientation, and the terms areused interchangeably. For convenience of description except as noted,the apparatus is described as oriented in the collection orientation. Assuch, relational references such as to top, bottom, up, down, above,below, elevations, vertical, horizontal and the like are in relation tothe apparatus as oriented in the collection orientation. The apparatusmay be configured such that the apparatus may be stably supported in thecollection orientation. For example, the apparatus may have a baseconfigured for interfacing with a flat, substantially horizontal surface(e.g., counter top or table top) to stably support the apparatus in thecollection orientation, or may be held in a mounting structure thatmaintains the apparatus in the collection orientation. Although such anorientation is referred to as a “collection” orientation it should beappreciated that use of the apparatus is not limited to being orientedonly in the collection orientation or that only human biologicalmaterial collection may be performed while the apparatus is oriented inthe collection orientation. The apparatus may be advantageouslyconfigured to permit performance of many different operations with theapparatus when the apparatus is oriented in the collection orientation.The apparatus is described with primary reference to processing humanbiological material comprising adipose, but the apparatus may be usedfor processing other biological material not comprising adipose.

The apparatus of the first aspect may be used in a variety of processingapplications involving adipose or other human biological material. Theapparatus may, for example, be used for preparation of concentrated orseparated portions of the collected human biological material, forexample to produce a stromal vascular fraction rich in leuko stromalvascular cells, including stem cells, derived from adipose tissue. Asanother example, the apparatus may be used for preparation of a fatgraft comprising adipose. The apparatus has a design that accommodatesretention of a target material (e.g., leuko stromal vascular cells oradipose) in a single container from collection through preparation of aconcentrate product containing the target material. By target material,it is meant some component or components from or some portion orportions of collected human biological material of interest for recoveryfollowing processing in the apparatus, such as recovery in aconcentrated or modified form relative to the collected human biologicalmaterial (e.g., stromal vascular fraction concentrate rich in stem cellsand other leuko stromal vascular cells, cleaned adipose-containingfraction for fat grafting applications).

The apparatus of the first aspect includes a container having aninternal containment volume, with the internal containment volumecomprising a tissue retention volume, a filtrate volume, a collectionvolume, and a tapered portion. The tissue retention volume and filtratevolume are separated by a filter. The collection volume is within thefiltrate volume (i.e., is a part of the filtrate volume) and has abottom elevation corresponding to a bottom elevation of the filtratevolume and a top elevation that is lower than the bottom elevation ofthe tissue retention volume. The tapered portion tapers in a downwarddirection with at least a portion of the tapered portion being locatedbelow (at a lower elevation than) the bottom elevation of the tissueretention volume. The apparatus includes an inlet port in fluidcommunication with the tissue retention volume and configured forintroducing extracted biological material directly into the tissueretention volume, such as during a lipoplasty procedure. The apparatusincludes a suction port that is in fluid communication with the filtratevolume and provides access to the filtrate volume for suctioning fromthe filtrate volume material that may pass through the filter from thetissue retention volume to the filtrate volume, for example biologicalfluids that may separate from biological material introduced into thetissue retention volume.

As noted, the tissue retention volume and the filtrate volume of theapparatus of the first aspect are separated by the filter. By being“separated” by the filter it is meant that the filter forms at least aportion of the defining physical separation between the tissue retentionvolume and the filtrate volume. As will be appreciated, the filteraccommodates movement of fluid and undersize non-fluid material (e.g.,liberated undersize cells, tissue fragments, etc.) between the tissueretention volume and the filtrate volume, while retaining oversizematerial within the tissue retention volume. In some applications (e.g.,preparing a stromal vascular fraction concentrate), target material maycomprise biological material that during processing in the apparatuspasses through the filter and collects in the collection volume. Asdiscussed below, the apparatus may be configured to be received in acentrifuge for centrifuging the apparatus, and such centrifuging mayassist to concentrate such target material in the collection volume. Inother applications (e.g., preparing a fat graft), target material (e.g.,adipose) may be retained within the tissue retention volume followingprocessing.

The apparatus may be used during multiple processing steps to prepare,for example, a stromal vascular fraction concentrate (e.g., concentraterich in leuko stromal vascular cells) from human biological materialcomprising adipose or a fat graft containing adipose, without the needto transfer a target material being processed between differentcontainers for different processing steps. The apparatus may be usedinitially to collect the human biological material (e.g. tissue andfluids) during a lipoplasty procedure or other tissue extractionprocedure, or tissue that has already been extracted in anotherprocedure may be introduced into the apparatus for processing. Theapparatus may be portable and easily transportable between locationswhere collection or different processing operations may be conducted.

A number of feature refinements and additional features are applicableto the first aspect of the invention. These feature refinements andadditional features may be used individually or in any combination. Assuch, each of the following features may be, but are not required to be,used with any other feature or combination of the first aspect.

The apparatus may include an extraction port in fluid communication withthe internal containment volume and configured for removing processedbiological material from the internal containment volume. Any or all ofthe inlet port, the suction port and the extraction port may beconfigured for access therethrough from above the container into theinternal containment volume. The extraction port may be located above aportion of the filter, so that the advancing tip of a hypodermic needlepierces the filter when the tip of the hypodermic needle is advancedfrom the extraction port into the collection volume. The collectionvolume may include a nadir and the extraction port may be positionedabove the nadir so that the tip of a hypodermic needle inserted throughthe extraction port may be advanced vertically downward to the vicinityof the nadir of the collection volume.

The apparatus may include a mixing device disposed at least in partwithin the tissue retention volume for mixing contents within the tissueretention volume. The mixing device may comprise a rotatable shaftextending from outside of the internal containment volume to inside ofthe internal containment volume. The shaft may be made of a polymeric ormetallic material of construction. A preferred material of constructionfor the shaft is stainless steel, for example 303, 304 or 316 stainlesssteel. If a polymeric material of construction is used, it shouldpreferably be high-strength, for example an Ultem™ resin compositionavailable from Saudi Basic Industries Corp. (SABIC). The shaft maycomprise at the top a tapered receptacle adapted to mate with a taperedsyringe tip. The tapered receptacle may be fitted with an o-ring to sealagainst the tapered syringe tip when inserted into the taperedreceptacle, thereby permitting a good suction to be applied by thesyringe through the lumen to extract material from the tissue retentionvolume. The shaft may comprise a handle interface outside of theinternal containment volume. The apparatus may further include a handleinterfaced to the handle interface, wherein rotating the handle causesthe shaft to rotate, thereby operating the mixing device. The handle maybe removably interfaced to the handle interface, to permit the handle toconveniently be connected with the handle interface to operate themixing device when appropriate and to conveniently be removed from thehandle interface to permit access to a top of the apparatus withoutinterference from the handle. The apparatus may include a lumen thatextends through the shaft and has a proximal end located outside of theinternal containment volume and a distal end located within the internalcontainment volume. Such a lumen may provide access from outside of theinternal containment volume to inside of the internal containmentvolume. A removable plug for sealing the lumen may be disposed in theproximal end of the lumen. The shaft may be rotatable about an axis thatextends through the collection volume. The lumen may be aligned with theaxis. The lumen may provide access to the collection volume foraspiration of material therefrom and/or injection of material thereto.

The apparatus may be configured for advancing a hypodermic needlethrough a lumen and out of the distal end of the lumen to access thecollection volume with an advancing tip of the hypodermic needle. Thedistal end of the lumen may be located in the tissue retention volumeabove a portion of the filter, so that the advancing tip of thehypodermic needle may pierce and pass through the filter when the tip ofthe hypodermic needle exits the distal end of the lumen and is advancedfrom the distal end of the lumen into the collection volume. Thecollection volume may include a nadir, and an axis of the lumen may bealigned so that the tip of a hypodermic needle exiting the distal end ofthe lumen may be advanced to the vicinity of the nadir of the collectionvolume. The hypodermic needle may thus access the collection volume topermit injection of material into and/or aspiration of material from thecollection volume (e.g., aspiration of stromal vascular fractionconcentrate or other processed biological material collecting in thecollection volume during processing). The apparatus may be designed forsingle-use, and piercing the filter with a hypodermic needle maybeneficially provide a safety mechanism for preventing reuse, and risksassociated therewith, by damaging the filter in a way that renders thefilter unsatisfactory for reuse.

The mixing device, or mixer, may comprise at one or more mixing membersdisposed in the tissue retention volume and connected with the shaft,wherein the mixing member moves through the tissue retention volume whenthe shaft is rotated. For example, a mixing member may be in the form ofimpeller blades, paddles or arms that agitate and mix components withinthe tissue retention volume when the shaft is rotated. At least aportion of the tissue retention volume may be within the tapered portionof the internal containment volume (with at least a portion of thetapered portion being located above a bottom elevation of the tissueretention volume), and at least a portion of one or more such mixingmember may be disposed within the tapered portion of the internalcontainment volume. The mixing device may include a filter contactmember that moves when the shaft is rotated and movably contacts thefilter. The filter contact member may be part of or separate from such amixing member. The filter contact member contacts the filter at leastperiodically, and may contact the filter continuously when the shaft isrotated. The filter contact member may advantageously deform the filterwhen it moveably contacts the filter, promoting dislodgment of materialfrom the filter to help prevent filter clogging.

As noted, the suction port is in fluid communication with the filtratevolume. By the suction port being in fluid communication with thefiltrate volume, it is meant that the suction port is fluidly connecteddirectly to the filtrate volume, and not indirectly through the tissueretention volume and the filter. The fluid communication may be providedby a dedicated conduit extending from the suction port to a desiredlocation within the filtrate volume where it is desired to apply suctiondirectly to the filtrate volume. The suction port may be in fluidcommunication with the tapered portion of the internal containmentvolume through a conduit providing fluid communication from the suctionport to a location within the filtrate volume that is also within thetapered portion of the internal containment volume. The conduit mayextend through the filtrate volume from adjacent the suction port tosuch a location within the filtrate volume. The suction port may belocated above the tapered portion of the internal containment volume.The suction port may be configured for access through the suction portfrom above the container. The suction port may be configured forconnection to a vacuum system to suction material from the filtratevolume, such as material that passes through the filter from the tissueretention volume to the filtrate volume.

The apparatus may include multiple suction ports. For example, theapparatus may include a first suction port as described in the precedingparagraph that is in fluid communication with a first location in thefiltrate volume within the tapered portion of the internal containmentvolume through a first conduit, and the apparatus may include a secondsuction port through which components passing through the filter fromthe tissue retention volume to the filtrate volume may be suctioned fromthe filtrate volume through a second conduit extending from the secondsuction port to a second location within the filtrate volume. The secondconduit may be configured to permit adjustment of the elevation of thesecond location within the filtrate volume. The second conduit may betranslatable through the second suction port to adjust the elevation ofthe second location within the filtrate volume. The second conduit maybe configured so that at any extent of such adjustment of the secondlocation, the second location will always be at a higher elevationwithin the filtrate volume than the first location, which may be fixed.The second conduit may be configured to permit adjustment of theposition of the second location within the filtrate volume at differentelevations above the tapered portion of the filtrate volume. The secondconduit may be configured to permit positioning the second location atan elevation corresponding with an interface between the tapered portionof the filtrate volume and a portion of the filtrate volume locatedabove the tapered portion, which may be the lowest elevation to whichadjustment is permitted. The second suction port may be configured foraccess through the second suction from above the container.

Any one or more of the inlet port, the suction port of other portsproviding access to the internal containment volume may be configuredfor access through the port from above. In this way, access through eachsuch port may be conveniently from above the apparatus, providing asignificant advantage to a user of the apparatus in that such a user mayfocus all access manipulations from above the apparatus while theapparatus is in a normal position in the collective orientation, forexample with the apparatus freestanding on a flat work surface such as atable or counter. Complexities associated with access from the side orfrom below may be avoided, including the complexity of sealing andproviding access into side or bottom access ports against a positivefluid head that may be present in the container and the complexity ofawkward side of bottom interactions by users. Although such access fromabove the container may be at some angle relative to vertical, in apreferred implementation the access through such port is in a verticaldirection from above the container. In one preferred implementation, allaccess to the internal containment volume may be through access portswherein each such access port (e.g., inlet port, suction port,extraction port, other ports) is configured for access through theaccess port only from above the container. In another preferredimplementation, all access ports may be configured for access througheach such access port in a vertical direction from above the container.

The tapered portion may have a cross-sectional area that tapers, orreduces in size, in a direction toward the bottom of the collectionvolume. The tapered portion of the containment volume helps to directand concentrate target dense material (e.g., dense cells, stromalvascular fraction) toward and into the collection volume. The taperedportion of the containment volume may have a conical shape or any othershape with a cross-sectional area that tapers to reduce in size in adirection toward the bottom of the collection volume. In variousimplementations, at least a part of the tapered portion may be locatedabove the collection volume.

The container may have a self-supporting structure, with the containerhaving rigid walls or rigid structural supports to maintain thecontainer in a particular configuration. The container may be made ofany suitable material or materials of construction. The container may bemade of one or more plastic composition. The container may havetransparent walls. A preferred material of construction for thecontainer is a clear polymeric material, such as for example a clarifiedpolypropylene composition. Clarified polypropylene compositions providelow cellular adhesion and reasonable clarity. The container may becomprised of multiple pieces, which may all be made out of the samematerial of construction or one or more of such pieces may be made of adifferent material of construction.

The container may comprise a fluid containment shell with an internalcavity portion forming at least a part of the internal containmentvolume. The internal cavity portion may be open to above. The containermay include a lid attached to the shell and disposed to cover from abovethe internal cavity portion. One or more than one of the suction portthe inlet port or other access port may pass through the lid. In onepreferred implementation, all access into the internal containmentvolume may be only through one or more openings, or ports, passingthrough the lid. The filter may be suspended from the lid. The mixingdevice may be supported by the lid and extend vertically downward fromthe lid into the tissue retention volume. The apparatus may include aflow barrier skirt extending between 5 mm and 50 mm downward from thelid into the internal containment volume. The shell may comprise wallsaround the internal cavity portion except where the cavity portion isopen to above, and the apparatus may be configured with no access intothe internal containment volume through the walls of the shell. Theshell may comprise an upper portion having a first wall surface portiondefining a corresponding upper portion of the internal containmentvolume. Substantially all of the first wall surface portion may have asteep incline relative to horizontal, for example an incline of at least65°, preferably an incline of at least 75° and more preferably anincline of about 90° (vertical wall surface). The shell may include alower portion located below the upper portion and having a second wallsurface portion defining a corresponding lower portion of the internalcontainment volume, and the second wall surface portion may include atapered wall surface portion defining the tapered portion of theinternal containment volume. The tapered wall surface portion may have aless steep incline relative to horizontal than the first wall portion ofthe upper portion. The incline relative to horizontal of the taperedwall portion, or of the entire second wall portion when comprisedentirely of the tapered wall portion, may be in a range having an upperlimit of 70°, 65°, 60° or 65° and a lower limit of 20°, 25°, 30° or 35°,with one preferred range being form 30° to 60°. The tapered portion ofthe internal containment volume may occupy substantially the entirelower portion of the internal containment volume, and the second surfacemay be made up entirely or substantially entirely by the tapered wallsurface. At least a first portion of the filter may be disposed in theupper portion of the internal containment volume and a second portion ofthe filter may be disposed in the lower portion of the internalcontainment volume. The incline of each of the first wall surfaceportion, the second wall surface portion and the tapered wall surfaceportion need not be uniform, however all portions of the first wallsurface portion may preferably be at a steeper incline than the inclineof any portion of the tapered wall surface portion.

There are a number of advantages that may be available withconfigurations of the preceding paragraph including an upper portionhaving a steeper wall surface incline and a lower portion having a lesssteep wall surface incline. Such a structure advantageously accommodatesa larger proportion of the internal containment volume being allocatedto the tissue retention volume, with a larger portion of the tissueretention volume in the upper portion of the shell and a smaller portionof the tissue retention volume in a lower portion of the shell. Thetapered wall surface portion of the lower portion of the shell helps todirect fluid and other material in the filtrate volume toward the bottomof the filtrate volume for efficient collection and removal. Inapplications where target material is to be collected in the collectionvolume, the tapered wall surface also directs material toward thecollection volume, which may generally be located in a bottom portion ofthe filtrate volume. The apparatus may also be configured to becentrifugable, and centrifuging will tend to accelerate concentration ofa most dense fraction (e.g., stromal vascular fraction) in thecollection volume.

More generally, the tapered portion of the internal containment volumemay have a tapered portion nadir corresponding with a bottom elevationof the internal containment volume. The bottom elevation of thecollection volume may correspond with the bottom elevation of theinternal containment volume. Wall surfaces of the container defining thetapered portion of the internal containment volume may coverage at apoint at the tapered portion nadir. This is a particularly beneficialconfiguration, especially for applications when target material is to becollected in and removed from the collection volume in the vicinity ofthe tapered portion nadir.

The apparatus may be configured with a very convenient size from anumber of perspectives, and with efficient use of the internalcontainment volume to facilitate efficient collection of biologicalmaterial and versatility in post-collection processing. The apparatusmay be sized for convenient hand transportation, such as between alocation where human biological material may be collected to other,different locations, where various processing of collected material maybe carried out. The apparatus may also be sized for convenientmanipulation by a person.

For many applications, the apparatus may be sized and configured suchthat the internal containment volume has a volume in a range with alower limit of 100 cubic centimeters, 300 cubic centimeters, 500 cubiccentimeters, 600 cubic centimeters or 700 cubic centimeters and an upperlimit of 1500 cubic centimeters, 1300 cubic centimeters, 1100 cubiccentimeters, 1000 cubic centimeters, 900 cubic centimeters or 800 cubiccentimeters. One preferred range for many applications is for theinternal containment volume to be in a range of 700 cubic centimeters to1000 cubic centimeters. By internal containment volume, it meant thetotal internal volume contained within the walls defining the container,including volume that is occupied by internal hardware, such as forexample may be occupied by a mixing device, barrier member, suctionconduits, barrier skirt, etc. As will be appreciated, less than all ofthe internal containment volume will be available for processing withinthe internal containment volume.

The terms “available processing volume”, “useful volume” and “internalprocessing volume” are used interchangeably herein to refer to theportion of the internal containment volume that is effectively availableto receive and process human biological material and additives (e.g.wash liquid, enzyme solution, other additives) during use of theapparatus for collection of biological material or for post-collectionprocessing. This available processing volume is equal to the internalcontainment volume less portions of the internal containment volumeoccupied by hardware (e.g., mixing device, filter, skirt, suction tubes,barrier member, etc) and less unoccupied portions of the internalcontainment volume not effectively accessible for occupation bybiological material during collection operations or by biologicalmaterial or additives during post-collection processing. For example,the available processing volume may exclude a small volume at the top ofthe container that is above a bottom extension of the inlet port intothe internal containment volume. This small void space may be beneficialto permit space for fluid to slosh within the container when contents ofthe container may be internally mixed or externally agitated (e.g., by ashaker table). For many applications, the available processing volumemay be in a range having a lower limit of 75 cubic centimeters, 200cubic centimeters, 400 cubic centimeters, 500 cubic centimeters, 600cubic centimeters, 650 cubic centimeters or 700 cubic centimeters and anupper limit of 1300 cubic centimeters, 1100 cubic centimeters, 1000cubic centimeters, 900 cubic centimeters, 850 cubic centimeters, 800cubic centimeters or 750 cubic centimeters. In one preferredimplementation for many applications, the available processing volumemay be in a range of from 700 cubic centimeters to 850 cubiccentimeters.

Advantageously, the apparatus may be configured so that a large portionof the available processing volume is within the tissue retentionvolume, while still permitting a high level of performance for variousprocessing operations. The tissue retention volume may comprise at least60 percent, at least 65 percent or at least 70 percent of the availableprocessing volume with the container. Often, the tissue retention volumewill comprise not more than 95 percent, not more than 90 percent or notmore than 85 percent of the available processing volume. For manypreferred implementations, the tissue retention volume may comprise aportion of the available processing volume that is at least 400 cubiccentimeters, at least 500 cubic centimeters, at least 600 centimeters orat least 650 cubic centimeters. The apparatus may advantageously beconfigured with only a small portion of the available processing volumeoccupied by the collection volume, located below the filter. Forexample, the collection volume may comprise no more than 10 percent, nomore than 7 percent or no more than 5 percent of the availableprocessing volume.

For many preferred implementations the collection volume may be nolarger than 75 cubic centimeters, no larger than 50 cubic centimeters,no larger than 30 cubic centimeters or no larger than 20 cubiccentimeters. The collection volume may often be at least 1 cubiccentimeter, at least 2 cubic centimeters or at least 3 cubiccentimeters. In one preferred implementation, the collection volume maybe in a range of from 10 cubic centimeters to 30 cubic centimeters.Typically, the entire collection volume will make up part of theavailable processing volume.

The apparatus may be sized and configured to be containable within arelatively small envelope volume which may be particularly advantageousgiven the relatively large internal containment volume, availableprocessing volume and tissue retention volume that may be provided inthe container. For some preferred implementations, the apparatus may besized and configured to be containable within a first envelope volumedefined by a rectangular cuboid having a length dimension of no morethan 16 centimeters, a depth dimension of no more than centimeters and aheight dimension of no more than 18 centimeters. However, the apparatusmay be sized and configured to have some minimum size, for example as afunction of a desired size of internal containment volume, availableprocessing volume or tissue retention volume.

For some preferred implementations, the apparatus may have a size andconfiguration such that the apparatus may not be containable within asecond envelope volume defined by a rectangular cuboid having any one ormore of a length dimension, depth dimension or height dimension that issmaller than 10 centimeters, i.e., the apparatus would not fit withinany rectangular cuboid smaller than 10×10×10 centimeters.

The filter may be of any appropriate filter media design. The filter maybe any porous structure with openings sized to make a desiredseparation. The filter may be in the form of a mesh filter bag disposedwithin the internal containment volume, and that separates the internalcontainment volume between the tissue retention volume and the filtratevolume located on opposite sides of the filter bag. The filter may be arigid mesh screen. In some implementations, the filter may have aseparation size in a range having a lower limit of 70 microns, 100microns, 150 microns, at 175 microns, 200 microns, 300 microns or 400microns and an upper limit of 800 microns, 700 microns, 600 microns, 500microns, 475 microns, 450 microns, 425 microns, 400 microns, 350microns, 300 microns or 250 microns; provided that the upper limit islarger than the lower limit. Leuko stromal vascular cells will easilypass through a 200 micron filter, however a somewhat larger filter sizemay be advantageous to promote recovery of most or substantially all ofthe leuko stromal vascular cells in the filtrate volume. Smaller sizefilters may plug to a degree that significantly reduces cell yield interms of cell collection in and recovery from the filtrate volume. Inone implementation, the filter may have a separation size in a rangehaving a lower limit of 70 microns and preferably 80 microns and anupper limit of 125 microns, preferably 110 microns and more preferably100 microns. By separation size, it is meant the size at which thefilter effects separation between particles passing through andparticles rejected by the filter during normal operation. The separationsize may be determined by the size of openings provided in a surfacefilter, such as the mesh size of a mesh bag filter or of a rigid meshscreen filter.

In one preferred implementation, the filter may be a mesh filter. With amesh filter, the separation size will be the size of the mesh openings.In one preferred implementation, whether or not the filter is a meshfilter, the separation size for the filter, and the size of meshopenings when a mesh filter is used, may be in a range as describedabove for separation size. In some implementations, the size of the meshopenings may be in a range having a lower limit of 70 microns, 80microns, 90 microns, 100 microns, 125 microns or 150 microns and havingan upper limit of 400 microns, 350 microns, 300 microns or 250 microns.One range for some applications is from 150 microns to 250 microns,including for preparation of a fat graft or preparation of a stromalvascular fraction concentrate. The mesh filter may be of a flexible or arigid mesh material. In a preferred implementation, the filter may bemade of mesh material, more preferably a nylon mesh material. The filterneed not be continuous, and may be comprised of discrete filter areasdisposed at different locations between the tissue retention volume andthe filtrate volume. Alternatively, the filter may be comprised of asingle continuous filter area. The filter defines at least part of thephysical separation between the tissue retention volume and the filtratevolume; it need not define all of the physical separation between theissue retention volume and the filtrate volume. For example, there maybe internal walls defining at least a part of the physical separationbetween the tissue retention volume and the filtrate volume, with anexample being a skirt barrier that may be disposed at the top of theinternal containment volume and that may define a separation between thetissue retention volume and the filtrate volume in an upper portion ofthe internal containment volume. Another example may be a barrier memberthat blocks access from the tissue retention volume into the collectionvolume portion of the filtrate volume. As another example, the filtermay include filter areas supported by a frame, with structural membersof the frame defining a part of the physical separation between thetissue retention volume and the filtrate volume. In a preferredimplementation, the portion of the physical separation between thetissue retention volume and the filtrate volume that is provided by thefilter should generally be large to provide as much filter surface areaas reasonably possible.

The apparatus may be configured to be received by a centrifuge forcentrifuging. For example, the apparatus may be conveniently sized andconfigured to be received within a centrifuge bucket, and preferably ofa commercially available centrifuge. For example, the apparatus mayadvantageously be sized and configured to fit within a bucket of abucket assembly for a Sorvall ST-40 centrifuge. Two or more of theapparatus may be centrifuged simultaneously in a centrifuge. In onepreferred implementation, the apparatus may be sized and configured sothat two of the apparatus may be simultaneously centrifuged together ina dual bucket assembly, wherein each apparatus counterbalances the otherapparatus during centrifuging, for efficient processing. Alternativelyone apparatus could be processed within a blank weight as acounterbalance. The apparatus may be received in a centrifuge bucketwith the bottom of the apparatus adjacent to and facing the bottom ofthe bucket.

Configuring the apparatus for centrifuging may be particularlyadvantageous for applications when target material is to be collected inthe collection volume, such as for collecting a stromal vascularfraction concentrate in the collection volume. The collection volume isadvantageously positioned in the bottom portion of the internalcontainment volume where the most dense materials will collect duringcentrifuging.

The apparatus may include an extraction port in direct fluidcommunication with the filtrate volume and through which material isremovable from the filtrate volume separate from the suction port. In apreferred implementation, access through the extraction port is in avertical direction from above the container, for example with theextraction port passing through the top of the container. Although not apreferred implementation, in various applications the apparatus mayinclude an extraction port that is adjacent a bottom elevation of thefiltrate volume and configured for access to the collection volume.

The apparatus may be configured for the addition of additives to theinternal containment volume, and in particular directly into the tissueretention volume. As used herein, such an additive is any material addedto the internal containment volume other than the human biologicaltissue comprising adipose, such as from a lipoplasty procedure, to beprocessed in the apparatus. Such an additive may be added for example toaid processing within the apparatus or to become part of a compositionincluding target material to be recovered from the apparatus forfollowing processing. Examples of some additives to aid processing mayinclude wash liquid, enzymes or surfactants. Examples of some additivesthat may become part of a fat graft composition include hormones (e.g.,human growth hormone, insulin), buffers (e.g., sodium bicarbonate) andcells (e.g., bone marrow-sourced stem cells, cultured adipose-sourcedstem cells, stromal vascular cells from adipose tissue). Such additivesmay be added to the tissue retention volume through the inlet port.

The apparatus may further include a second port in fluid communicationwith the tissue retention volume for introducing an additive directlyinto the tissue retention volume or for removing material (e.g., adiposetissue) from the tissue retention volume. The second port may be smallerthan the inlet port through which the human biological material to beprocessed (e.g., from a lipoplasty procedure) is introduced into thetissue retention volume. The second port, which may be referred to as anauxiliary port or as an additive port, may be conveniently sized andconfigured for insertion therethrough of a hypodermic needle from whichan additive material may be ejected from the needle into the tissueretention volume or through which material may be removed from thetissue retention volume. This auxiliary port may be configured to make aluer connection with a syringe, in which case a syringe may be attachedto the auxiliary port and the container tipped (inclined) in thedirection of the auxiliary port to facilitate suctioning material fromthe tissue retention volume directly into the syringe without the needfor a needle or cannula, and may be drawn into a syringe in fluidcommunication with the hollow member.

The apparatus may include human biological material, which may includetarget material from human biological material originally collected inthe apparatus disposed within the internal containment volume (e.g., theapparatus during some stage of use). The apparatus may includeadipose-containing material (e.g., collected material including adiposetissue) in the tissue retention volume. The apparatus may include in thetissue retention volume an adipose-containing fat graft composition,including any desired additives, ready to be withdrawn from the tissueretention volume and used in a fat graft procedure. The apparatus mayinclude a stromal vascular fraction concentrate disposed in thecollection volume.

The apparatus may be packaged within a hermetic enclosure, for exampleas packaged for transportation and storage prior to use. The apparatusmay be sterilized prior to packaging and maintained in a sterileenvironment within the hermetic enclosure at least until the apparatusis removed from the hermetic enclosure for use. The apparatus may bedesigned for a single use following removal from the hermetic enclosure.After such single use, the apparatus may be disposed of in anappropriate manner.

The apparatus may be fluidly connected through the inlet port to apre-filter. The fluid connection may be through a conduit that fluidlyconnects the inlet port to a downstream side of a pre-filter unitcomprising the pre-filter. The pre-filter unit may comprise a housingwith the pre-filter disposed within the housing, and with an inlet on anupstream side of the pre-filter and an outlet on downstream side of thepre-filter. The upstream side of the pre-filter may be in fluidcommunication with a lipoplasty cannula to receive extracted biologicalmaterial during a lipoplasty procedure and to pre-filter the biologicalmaterial prior to delivery to the inlet port of the apparatus, forexample to remove some collagen from the biological material upstream ofthe apparatus. The pre-filter may be provided with my convenientseparation size, for example in a range having a lower limit of 0.5millimeter or 1 millimeter to an upper limit of 5 millimeters, 3millimeters or 2 millimeters. The pre-filter may comprise a mesh screenwith mesh openings sized, for example, in such a range.

A second aspect of the invention is provided by a method of processingadipose tissue to concentrate leuko stromal vascular cells associatedwith the adipose tissue. The method combines particular processing incombination with a portable container to address significant designconstraints associated with the use of portable containers formulti-step processing of adipose tissue. The method of the second aspectincludes multi-step processing within a portable container having afilter inside the container. The multi-step processing includes washingthe adipose tissue within the container to remove contaminants from theadipose tissue. The washing includes multiple wash cycles, with eachwash cycle comprising: adding a volume of aqueous wash liquid to thecontainer to contact the adipose tissue within the container; mixing thewash liquid and the adipose tissue in the container; and removing atleast a majority of the wash liquid with contaminants from the containeron a first side of the filter and retaining at least most of the adiposetissue in the container disposed on a second side of the filter. Themethod includes digesting adipose tissue within the container. Thedigesting is performed after the washing. The digesting comprises addingto the container of volume of digestion medium comprising a collagenaseenzyme solution to contact washed adipose tissue in the containerfollowing the washing, wherein the volume ratio of the volume of digestmedium to volume of adipose tissue within the container is in a range offrom 0.6:1 to 2:1 and wherein the digestion medium provides from 150 to300 collagen digestion units (CDU) per milliliter of catalytic volume,and wherein the catalytic volume is the total of the volume of digestionmedium and the volume of adipose tissue within the container. Thedigesting also comprises, after adding the volume of digestion medium,permitting enzymatic digestion within the container for a retention timein a range of from 20 minutes to 50 minutes while the container isdisposed in a temperature controlled environment with the temperaturecontrolled environment maintained within a temperature range of from 32°C. to 38° C. and with at least occasional agitation of contents withinthe container. The method also includes, not later than 50 minutesfollowing adding of the volume of digestion medium, adding a stoppingreagent to the container to stop enzymatic activity within thecontainer. The method also comprises disposing the container in acentrifuge and centrifuging the container in the centrifuge deformdensity-separated phases within the container. The density-separatedphases include lower-density material phases and a higher-density pelletphase comprising leuko stromal vascular cells. After the centrifuging,the method includes removing the container from the centrifuge andremoving the lower-density material phases from the container whileretaining the pellet phase within the container.

The method particularly addresses processing within the constrainedcontext of multiple-step processing within a single portable container.The method may permit effective processing within such a portablecontainer in a manner to address inherent equipment and processingdesign problems associated with multi-step processing in portablecontainers and without excessive losses of cell viability or physicallosses of cells to adherence to equipment and container surfaces insidethe container.

A number of feature refinements and additional features are applicableto the second aspect of the invention. These feature refinements andadditional features may be used individually or in any combination. Assuch, each of the following features may be, but are not required to be,used with any other feature or combination of the second aspect or thefirst aspect of the invention.

In preferred implementations, the portable container may be a containerof an apparatus of the first aspect of the invention. The first side ofthe filter within the container may be the filtrate volume and thesecond side of the filter in the container may be the tissue retentionvolume of a container of an apparatus of the first aspect of theinvention. Alternatively, the portable container may be other than acontainer of the apparatus according to the first aspect of theinvention.

The step of removing the lower-density material phases may includeremoving such lower-density material phases from the container insequence of increasing density, which may include suctioning thelower-density material phases from the container through an open end ofa suction conduit disposed in the container. Preferably, such an openend of a suction conduit may be disposed in the container not directlyabove the pellet phase, to reduce the possibility that suction createdin the container would structurally disrupt the pellet phase. In somepreferred implementations, the pellet phase remains in place andstationary, relative to the container, while the lower-density materialphases are removed. During the centrifuging, the pellet phase may format a location within the container adjacent a bottom of the internalcontainment volume, and the pellet phase may remain at the locationduring the removing the lower-density material phases. In someimplementations, removing the lower-density material phases may includetipping the container during suctioning of lower-density material phasesto promote flow of at least a final suction portion of the lower-densitymaterial phases within the container laterally away from the pellet andtoward the open end of the suction conduit. The container may include acorner located lateral to the pellet phase, and which may be located atan elevation of the container that is higher than the bottom elevationof the pellet phase, or even higher than a top elevation of the pelletphase. The tipping may promote flow of fluid of the lower-densitymaterial phases laterally toward the corner for suctioning from thevicinity of the corner into the open end of the suction conduit. In somepreferred implementations, the lower-density material phases are removedthrough a top of the container.

The method may include one or more steps in addition to the steps notedabove. Any such additional step may be performed between any of thesteps noted above or may be performed prior to the washing or after theremoving of the lower-density material phases from the container.

The method may include introducing aqueous suspension liquid into thecontainer and dispersing cells of the pellet phase in the suspensionliquid, such as to form a dispersion of the cells in the suspensionliquid. The suspension liquid may be introduced into the portablecontainer apparatus after the lower-density material phases have beenremoved. The suspension liquid may be introduced at a volume in a rangehaving a lower limit of 1, 2, 3 or 5 milliliters and an upper limit of25, 20, 15 or 12 milliliters. A volume of suspension liquid of about 10milliliters may be used for many implementations. A volume ratio of thesuspension liquid to the volume of the pellet phase may be in a rangehaving a lower limit of 1:1, 2:1, 3:1 or 5:1 and an upper limit of 25:1,20:1, 15:1 or 12:1. A volume ratio of about 10:1 may be used in manyimplementations. After being dispersed in a suspension liquid, thesuspension liquid with dispersed cells may be removed from thecontainer. Preferably at least most of the suspension liquid is removedfrom the container and more preferably substantially all of thesuspension liquid and substantially all of the cells from the pelletphase are removed from the container with the suspension liquid. Duringthe centrifuging, the pellet phase may form within the portablecontainer apparatus adjacent a bottom of the internal containment volumewhere the suspension liquid may mix with the pellet phase to form thesuspension. The suspension liquid and dispersed cells may be removedthrough a top of the container, even though suspension liquid anddispersed cells may be removed from a location adjacent a bottom of thecontainer. This suspension liquid and dispersed cell may be removedupward through a hollow member disposed downward into the container, forexample through a hollow needle or cannula, and may be drawn into asyringe in fluid communication with the hollow member. In someimplementations, such a hollow member may pierce and extend across thefilter.

The method may include removing leuko stromal vascular cells of thepellet phase from the container, which may include the use of asuspension liquid as noted above. At least a majority of the leukostromal vascular cells of the pellet phase may be removed from theportable container apparatus, and preferably all or almost all of theleuko stromal vascular cells of the pellet phase are removed from thecontainer.

The digestion medium may provide collagen digestion units (CDU) permilliliter of catalytic volume within a range that is narrower than therange listed above. Such a range may have a bottom limit of 150, 175 or200 CDU and an upper limit of 300, 275 or 250 CDU. In someimplementations, the digestion medium may provide about 225 CDU permillimeter of catalytic volume. In this regard, the catalytic volume isthe total volume of the digestion medium added to the container and thevolume of adipose tissue already disposed within the container when thedigestion medium was added. For example, if the volume of digestionmedium added to the container equals the volume of adipose tissuealready disposed within the container, then the digestion medium willneed to contain a concentration of collagenase enzyme that is twice aslarge as the desired concentration relative to the catalytic volume. Aswill be appreciated, the adipose tissue as collected will haveassociated contaminants, but in preferred applications with thoroughwashing, the adipose tissue should be cleaned of most contaminants sothat substantially all of the volume of material on the second side ofthe filter in the container will be adipose tissue.

The volume ratio of digestion medium to adipose tissue may be in anarrower range than that described above. Such a volume ratio may have alower limit of 0.6:1, 0.75:1 or 0.9:1 and may have an upper limit of2:1, 1.75:1, 1.5:1 or 1.25:1. For various implementations, the volumeratio of digestion medium to washed adipose tissue may be about 1:1.

The retention time during the digesting may be within a narrower rangethan that described above. The retention time may be in a range having alower limit of 20 minutes, 25 minutes or 30 minutes and an upper limitof 50 minutes, 45 minutes or 40 minutes. For various implementations,the retention time may be about 35 minutes.

The digesting may include continuous agitation of the contents duringsome portion or substantially all of the retention time. The agitationmay include mixing, periodically or continuously, with a rotatable mixerdisposed within the container. The agitation may include periodic orcontinuous movement of the container to cause agitation of contentswithin the container. The agitation may include shaking the container,such as on a warmer-shaker. The temperature controlled environment maybe provided by a warmer-shaker.

Temperature control may be implemented at various points in theprocessing of the method. The digestion medium when added to thecontainer may be within a temperature range having a lower limit 32° C.,33° C., 34° C. or 35° C. and an upper limit of 38° C. or 37° C. Thetemperature within the temperature controlled environment may bemaintained in a narrower range than that stated above. The temperaturecontrolled environment may be maintained within a temperature rangehaving a lower limit of 32° C., 33° C., 34° C. or 35° C. and an upperlimit of 38° C. or 37° C. The wash liquid, when added to the container,may be within a temperature range having a lower limit of 32° C., 33°C., 34° C. or 35° C. and an upper limit of 38° C. or 37° C.

For each wash cycle, a volume ratio of wash liquid addition may becontrolled. The volume ratio of wash liquid addition refers to a volumeratio of the volume of wash liquid to a volume of adipose tissue withinthe container to which the wash liquid is being added during the washcycle. The volume ratio of wash liquid addition may be in a range havinga lower limit of 0.5:1, 0.7:1 or 0.8:1 and an upper limit of 4:1, 3:1,2:1 or 1.5:1. For many implementations, the volume ratio of wash liquidaddition may be about 1:1. A cumulative volume ratio of wash solutionaddition may be at least 2:1, or at least 3:1. The cumulative volumeratio of wash solution addition refers to a sum of the volume ratios forall of the wash cycles.

The washing may include more than two wash cycles. In someimplementations, the washing may comprise at least three wash cycles.For many implementations, three wash cycles may be sufficient, while forother implementations, two wash cycles may be sufficient.

Each wash cycle may comprise removing wash liquid (preferably at least amajority of the wash liquid and more preferably substantially all of thewash liquid) by suctioning from the container on the first side of thefilter (from the filtrate volume). During such suctioning, the washliquid may be removed through a top of the container.

Mixing the wash liquid may include operating a rotatable mixer disposedin the container. The rotatable mixer may be manually operable, such asby a handle attached to a rotating shaft disposed through a top of thecontainer. The mixing may include manually (hand) manipulating such ahandle to manually rotate the mixer within the container. In variouspreferred implementations, such mixing may be performed followingaddition of the wash liquid, and preferably shortly following suchaddition, to thoroughly mix the wash liquid and the adipose tissue beingwashed. Such a rotatable mixer may also be used to mix the digestionmedium and the adipose tissue following addition of the digestion mediumto the container, and preferably shortly after such addition, tothoroughly mix the digestion medium and washed adipose tissue to bedigested.

The wash liquid used during the washing may but need not be of the samecomposition for each wash cycle. The wash liquid may include one or moreadditives. For example the wash liquid for one of more of the washcycles may include one or more than one of an anti-clotting agent, anantibiotic and an antifungal. In some preferred implementations, for atleast one wash cycle, the wash liquid includes at least one of ananti-clotting agent, an antibiotic or an antifungal. In otherimplementations, for at least one wash cycle, the wash liquid includesan anti-clotting agent, an antibiotic and an antifungal. One preferredexample for an anti-clotting agent is heparin.

The adding of a stopping reagent to the container may be performedwithin a narrower time period than that described above. The stoppingreagent may be added within a time period not later than 45 minutesfollowing adding the volume of digestion medium, not more than 40minutes following adding the volume of digestion medium or not more than35 minutes following adding of the volume of digestion medium. Thestopping reagent may comprise human albumin. The stopping medium may beadded in an amount sufficient to substantially stop enzymatic activitywithin the container. The stopping reagent may preferably be addedbefore the centrifuging of the container following the digesting.

The container may be conveniently transported between differentlocations for performance of different processing at the differentlocations, and preferably may be manually transported by being carriedby a person. For example, the temperature controlled environment may belocated at one location and the centrifuge may be located at a differentlocation, and the method may comprise after the retention time in thetemperature controlled environment, transporting the container from thatlocation to the location of the centrifuge for performance of thecentrifuging. As another example, one or more wash cycles may occur atyet a different location, and the method may comprise transporting thecontainer from the location of a wash cycle to the location of thetemperature controlled environment. By transporting the container fromone location to another it is meant that the container, along withcontents of the container, are physically moved from one location to theother location, whether or not there are intermediate stops along theway.

The container may have an access orientation, also referred to herein asa collection orientation, which may be a free-standing orientation. Whenthe container is in the access orientation, all access into thecontainer may in various implementations be through one or more portsextending through the top (e.g., through a lid) of the container andaccessible from above the top of the container. Having all access intothe container from above the container facilitates convenient additionand removal of materials from the container, without requiring specialsuspension or retention of the container and without access from theside that may be more susceptible to moving or tipping the container.When in the access orientation, the container may be supported by a basethat maintains the container in a stable, free-standing condition.

The container may include volume gradation markings on an exterior sideof the container, with the gradation markings indicating the quantity ofvolume occupied by a tissue retention volume (e.g., on a second side ofthe filter) within the container up to different elevations within thecontainer, such as when the container is positioned an accessorientation. The gradation markings permit direct visual measurement ofthe quantity of the volume of material (e.g., adipose tissue) disposedwithin the tissue retention volume. This makes it convenient for someoneusing the container to quickly identify the volume of tissue disposedwithin the tissue retention volume, and to quickly determine quantitiesof wash liquid or digestion medium to be added for washing or digestingoperations. When the container includes such gradation markings, thewall of the container with the gradation markings preferably hassufficient transparency to permit visual observation of the level oftissue or other material disposed within the container.

The method permits convenient and controlled processing of significantquantities of adipose tissue in a convenient manner. The volume ofadipose tissue (including contaminants), disposed in the container onthe second side of the filter (e.g., within a filtrate volume) atcommencement of the washing may be in a range having a lower limit of50, 100, 150, 200 or 250 cubic centimeters and an upper limit of 700,600, 500 or 400 cubic centimeters.

It should be appreciated that when reference is made to “adipose tissue”or a volume thereof in relation to a method of the invention thereference may be to in-tact adipose tissue and any associatedcontaminants that are present with the in-tact tissue. Thesecontaminants come from the biological materials extracted from subjectsto obtain the adipose tissue. Contaminants that may be associated withthe adipose tissue include for example blood, free lipids, smallparticles and debris and other materials that may have been collectedwith the adipose tissue or result from degradation during tissuecollection or processing operations. The amounts of these contaminantswill generally be higher in unwashed adipose tissue at the commencementof washing operations and will generally be lower at the commencement ofdigesting operations, following the washing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in perspective a tissue collection and processingapparatus.

FIG. 2 shows the same tissue collection and processing apparatus as FIG.1 with some component parts shown in exploded view.

FIG. 3 shows in perspective a tissue collection and processing system.

FIG. 4 shows side and end sectional views of a tissue collection andprocessing apparatus having collected tissue disposed therein.

FIG. 5 shows a tissue collection and processing apparatus suspended by ahandle and from which material is being removed from the filtrate volumethrough an extraction port.

FIG. 6 shows the same tissue collection and processing apparatus asshown in FIG. 5 during removal of material from the filtrate volumethrough an extraction port.

FIG. 7 shows a tissue collection and processing apparatus suspended by ahandle and from which material is being removed from the filtrate volumethrough a suction tube inserted into a suction port.

FIG. 8 shows a tissue collection and processing apparatus being mountedon a warmer-shaker.

FIG. 9 shows a centrifuge with two tissue collection and processingapparatus received therein for centrifuge processing.

FIG. 10 shows a tissue collection and processing apparatus suspended bya handle and from which material is being removed from a collectionchamber through an extraction port.

FIG. 11 shows top, perspective, side and end views of another embodimentof a tissue collection and processing apparatus.

FIG. 12 shows another perspective view of the same tissue collection andprocessing apparatus as FIG. 11.

FIG. 13 shows the same tissue collection and processing apparatus asFIG. 12 with a shell removed.

FIG. 14 shows the same tissue collection and processing apparatus asFIG. 13 with a filter removed.

FIG. 15 illustrates various regions within the tissue collection andprocessing apparatus of FIG. 12.

FIG. 16 illustrates a needle inserted into a tissue collection andprocessing apparatus.

FIGS. 17A and 17B illustrate a translatable conduit in a tissuecollection and processing apparatus.

FIG. 18 illustrates a pre-filter and a tissue collection and processingapparatus.

FIGS. 19-22 illustrate various operations in a method of processingtissue within a tissue collection and processing apparatus.

FIG. 23 shows top, perspective, side and end views of another embodimentof a tissue collection and processing apparatus.

FIG. 24 shows an exploded view of the same tissue collection andprocessing apparatus as FIG. 23.

FIG. 25 is a generalized process block diagram of an embodiment of amethod of processing adipose tissue.

FIG. 26 is a generalized process block diagram of another embodiment ofa method of processing adipose tissue.

FIG. 27 illustrates transportability of a container to accommodateperforming different processing operations at different locations.

FIG. 28 shows a configuration of the tissue collection and processingapparatus of FIG. 12.

FIG. 29 shows another configuration of the tissue collection andprocessing apparatus of FIG. 12.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 1 shows one embodiment of a tissue collection and processingapparatus, designated as apparatus 100. FIG. 2 shows the same apparatus100 as in FIG. 1, but illustrated in an exploded view of some of thecomponents. As shown in FIGS. 1 and 2, the apparatus 100 has a mesh bagfilter 102 disposed within an internal containment volume of acontainer. The container is comprised of a rigid shell 104 and a rigidtop 106. The top 106 is sealed to the top of the shell 104. The shell104 may be made, for example, from a transparent plastic composition.The lid 106 may be made, for example, from a plastic composition.Disposed at the bottom of the shell 104 is a collection chamber 108 andan extraction port 110. The extraction port 110 comprises a sealingmechanism that is manipulable to extract material from inside thecollection chamber 108. The sealing mechanism may, for example, comprisea silicon rubber septum or diaphragm that may be penetrable by ahypodermic needle to extract material, or may comprise a valve.

The mesh filter bag 102 divides and separates the internal containmentvolume of the container into a tissue retention volume 112 disposedinside the mesh filter bag 102, and a filtrate volume 113 disposedwithin the shell 104 on the outside of the mesh filter bag 102. Thefiltrate volume 113 is that portion of the internal containment volumeinto which filtrate enters after passing through the mesh filter bag 102from the tissue retention volume 112.

An inlet port 114 in fluid communication with the tissue retentionvolume 112 through the top 106 is configured for introducing adiposetissue directly into the tissue retention volume during a lipoplastyprocedure, such as for example through a tissue transport conduit thatmay be attached to the inlet port 114 to conduit tissue from a patientduring such a procedure. An additional access port 116 in fluidcommunication through the top 106 with the tissue retention volume 112provides an additional route for introducing material into or removingmaterial from the tissue retention volume 112. A suction port 118 is influid communication through the top 106 with the filtrate volume 113 viaa conduit 120 extending from the suction port 118 to the vicinity of thetop of the collection chamber 108. The suction port 118 is configuredfor connection to a vacuum system, for example through connection of asuction conduit through which suction may be applied by a vacuum systemto suction from the filtrate volume material passing through the meshfilter bag 102 from the tissue retention volume 112 into the filtratevolume 113. The shell 104 has a tapered wall portion 122 that defines atapered portion of the internal containment volume, such that thecross-sectional area of the tapered portion of the internal containmentvolume tapers with a reducing cross-sectional area in a direction towardthe collection chamber 108. By tapering, it means that thecross-sectional area in a horizontal plane (assuming the apparatus 100is in an upright position as shown in FIG. 1) becomes smaller in acontinuous manner in the direction of the taper (e.g., a directionorthogonal to the horizontal plane). The collection chamber 108comprises a cylindrical volume located immediately below the taperedportion of the internal containment volume. The cross-sectional area ofthe internal containment volume at the bottom of the tapered portion 122approaches that of the cross-sectional area of the collection chamber108. “Collection chamber 108” is used to refer both to the downwardlyextending cylindrically-walled portion of the shell body 104 and thecylindrical portion of the filtrate volume disposed therein.

The shell 104 includes a base portion 123 configured to support theapparatus 100 in the upright position as shown in FIG. 1, for examplewhen the apparatus is resting on top of a hard flat surface, such as atable or shelf. For convenience, the side of the container adjacent thetop 106 is referred to as the top side of the container and the side ofthe container adjacent the extraction port 110 is referred to as thebottom side of the container.

The apparatus 100 comprises mixers 124 that have agitator arms 126 thatare rotatable to help mix contents within the internal containmentvolume, and in particular within the tissue retention volume 112. Themixers 124 may be driven by electrical power to rotate the agitator arms126, which power may be supplied, for example, by an external electricalpower source or by batteries disposed within the body of the mixers 124or elsewhere in the apparatus 100.

The apparatus 100 includes a retractable handle 128 to facilitatesuspending the apparatus 100 or for grasping and holding the apparatus100 by hand. As shown in FIG. 1, the handle 128 is in an extendedposition for use to grasp or suspend the apparatus 100. FIG. 2 shows thehandle 128 in a retracted position that is conveniently out of the wayso that the handle 128 does not interfere with access to the inlet port114, the access port 116 or the suction port 118 during use of theapparatus 100.

The apparatus 100 is designed to be portable, and is preferably portableby someone grasping the handle 128 and picking up the apparatus 100 bythe handle 128 by hand, preferably by using a single hand, to facilitateready transport of the apparatus 100, either while the internalcontainment volume is empty or with human tissue or components thereofdisposed within the internal containment volume.

Reference is now made to FIG. 3 showing a tissue collection andprocessing system 200 including the tissue collection and processingapparatus 100 of FIGS. 1 and 2 with an inlet port fluidly connected to atissue conduit 204. A suction port of the apparatus 100 is fluidlyconnected with a canister 206 via a suction conduit 208. The canister206 is fluidly connected with a vacuum system (not shown) through aconduit 210. For illustration purposes, the tissue collection andprocessing apparatus shown in FIG. 3 is the apparatus 100 of FIGS. 1 and2. However any other design could be used in such a tissue collectionand processing system, including any of the apparatus designs describedbelow. During operation of the tissue collection and processing system200, the tissue conduit 204 is conducting adipose tissue to the inletport of the apparatus 100 for introduction of the adipose tissue intothe tissue retention volume of the apparatus 100. Suction is applied tothe filtrate volume within the apparatus 100 by the vacuum systemthrough the conduit 210, the canister 206 and the suction conduit 208 toremove by suction from the filtrate volume material separating from theadipose tissue in the tissue retention volume of the apparatus 100 andpassing through the filter and into the filtrate volume of the apparatus100. Such material suctioned from the filtrate volume through thesuction conduit 208 may then be collected in the canister 206. Thecanister 206 may be a waste canister and the collected material may bewaste for appropriate disposal. For example, red blood cells mayseparate from adipose tissue during collection of the adipose tissue inthe apparatus 100 and such red blood cells passing through the filterwill be removed from the filtrate volume of the apparatus 100 by suctionvia the suction conduit 208.

In a method for processing tissue from a lipoplasty procedure, thetissue may be processed within a containment volume of a portable tissuecollection and processing apparatus to prepare within the apparatus aconcentrated product comprising at least one target component, or atleast one target material, from the tissue. The apparatus has a filterand a container having an internal containment volume, wherein theinternal containment volume comprises a tissue retention volume and afiltrate volume separated by the filter. The method may comprise:washing tissue in the containment volume with a wash liquid; after thewashing, digesting tissue within the containment volume; and after thedigestion, centrifuging the apparatus to prepare in the filtrate volumea concentrate product comprising at least one target component. Forexample the concentrate product may comprise, or may consist essentiallyof, stromal vascular fraction from adipose tissue, and a targetcomponent may be or comprise stem cells from adipose tissue. The methodmay also comprise one or more steps in addition to the washing,digesting and centrifuging. For example such an additional step mayoccur prior to the washing, between the washing and digesting, betweenthe digesting and centrifuging or after the centrifuging.

During the washing, the wash liquid may be added to the containmentvolume to contact tissue within the tissue retention volume and with atleast a portion, preferably a majority, and more preferably most, of thewash liquid passing through the filter into the filtrate volume. Thewash liquid may wash one or more component from the tissue whileretaining washed tissue in the tissue retention volume. The washedtissue may be retained in the tissue retention volume by the filter.Wash liquid passing into the filtrate volume may be removed from thefiltrate volume, along with any component or components washed from thetissue. After adding the wash liquid, an optional step of centrifugingthe apparatus may be performed. Such centrifuging may facilitate a highdegree of separation of the wash liquid from the tissue retained in thetissue retention volume. Next, the wash liquid may be removed from thefiltrate volume, for example by being suctioned through a suction portof the apparatus or by removal through an extraction port of theapparatus. The wash liquid may be an aqueous liquid, and may be orcomprise a saline solution, for example a phosphate buffer solution. Toensure thorough washing of the tissue, the washing may include multiplewash stages, with each stage comprising adding wash liquid to thecontainment volume to contact tissue within the tissue retention volumeand removing wash liquid from the filtrate volume.

During the digestion, an enzyme, such as for example collagenase, isadded to the containment volume to contact at least a portion,preferably a majority of, and more preferably all or substantially allof the washed tissue, within the tissue retention volume. The enzymeshould be of a type capable of breaking down at least a portion of thewashed tissue to an extent to release a target component, or material,in a form capable of passing through the filter and into the filtratevolume. After adding the enzyme, the digesting may comprise agitatingcontents of the containment volume of the apparatus for a time and at atemperature sufficient for the digestion to proceed to an extent tosignificantly release the target component, or material, in the desiredform capable of passing through the filter. The agitating may involveany method to agitate contents of the containment volume, including forexample one or both of: (a) shaking the apparatus to agitate thecontents within the apparatus and (b) mixing the contents within theapparatus, such as with one or more mixing device disposed within thecontainment volume and preferably disposed within the tissue retentionvolume. Shaking the apparatus may be accomplished by mounting theapparatus on a shaker, and preferably a warmer-shaker with a temperaturecontrol feature so that the apparatus and its contents may be maintainedat a controlled temperature, such as at or approximately at human bodytemperature.

Post-digestion centrifuging promotes separation of the target componentfrom the digested tissue and passage of the target component through thefilter for collection in the filtrate volume, such as for example tocollect within a collection chamber at the bottom of the apparatus, forexample a collection chamber such as shown in FIG. 1 or FIG. 2. Thecentrifuging causes a concentrate product to collect in the filtratevolume, and preferably in such a collection chamber. Multiple materialphases may collect within the filtrate volume, one or more of which orone or more portions of which, may comprise the desired concentrateproduct containing a target component. For example, a desiredconcentrate product may be higher-density pellet phase that is enrichedin leuko stromal vascular cells, and which may be disposed near thebottom of the internal containment volume with multiple lower-densitymaterial phases disposed above the pellet in the internal containmentvolume. Such a higher-density pellet is also referred to herein as astromal vascular fraction pellet.

Before the washing, the method may comprise collecting the tissue in theinternal containment volume of the apparatus. The collecting maycomprise conducting adipose tissue removed from a patient during alipoplasty procedure into the tissue retention volume through a tissueconduit fluidly connected with the apparatus during the lipoplastyprocedure. Such collection may be performed, for example, using a tissuecollection and processing system such as shown in FIG. 3. During thecollecting, fluid separating from the adipose tissue and passing throughthe filter into the filtrate volume may be immediately removed from thefiltrate volume by suctioning the fluid from the filtrate volume and outof the apparatus, for example to a collection canister such as thatshown in FIG. 3.

The method may comprise, after the centrifuging, removing theconcentrate product from the filtrate volume of the apparatus. Theconcentrate product may, for example, be removed from an apparatus suchas those illustrated in FIGS. 1 and 2 from the collection chamber at thebottom of the filtrate volume through the extraction port withmanipulation of the sealing mechanism. During the removing, theconcentrate product may be removed to and collected in the barrel of asyringe, or in the barrels of multiple syringes. The removing mayinclude selectively removing the concentrate product from the filtratevolume, to isolate the concentrate product from other material that mayhave collected in the filtrate volume during the centrifuging. To assistremoving the concentrate product, the concentrate product may be dilutedwith a dilution liquid to put the concentrated product in a dilute formthat is easier to remove from the filtrate volume. The dilution liquidis used to suspend material of the stromal vascular fraction pellet, andmay alternatively be referred to a suspension liquid or re-suspensionliquid. The dilution liquid may be an aqueous liquid. The dilutionliquid may be a saline solution, for example a phosphate buffersolution. Such dilution of the concentrate product may be particularlyuseful in the situation where the concentrate product collects in theform of a relatively hard pellet in the filtrate volume, which may bethe case for collection of stromal vascular fraction from adiposetissue. Selective removal of the concentrate product may includeseparating a material phase comprising the concentrate product from oneor more other material phase that collects in the filtrate volume. Forexample, a stromal vascular fraction may be a middle density phase, witha more dense phase and a less dense phase disposed on either side of thestromal vascular fraction. The more dense phase, which may collect atthe bottom of a collection chamber in the apparatus, may be rich in redblood cells. Selective removal of the stromal vascular fraction mayinclude first removing this red blood cell phase from the collectionchamber (e.g., into a syringe) and then removing the stromal vascularfraction from the collection chamber (e.g., into a different syringe).However, with thorough initial washing of tissue, such a layer rich inred blood cells may be kept sufficiently small in size that it need notbe removed separately from the stromal vascular fraction pallet, and maybe removed with the dilution liquid together with the stromal vascularfraction.

The apparatus used with the method for processing tissue is portable.The method may include transporting the apparatus, and tissue containedtherein, between locations where different processing procedures areperformed. For example, the apparatus may be located at one locationwhere collecting tissue is performed, while the washing and/or digestingmay be performed at a different location. In one implementation, thecontainment volume comprising tissue may be sealed following oneprocedure and the apparatus with the sealed containment volume may betransported to a different location for performance of a subsequentprocedure. For example, with the apparatus 100 shown in FIGS. 1 and 2,the apparatus may be sealed by capping the inlet port 114, access port116 and suction port 118 with sealing caps.

To further illustrate various features of the method for processingtissue, and apparatus, assemblies and systems that may be used duringthe method, reference is now made to FIGS. 4-10.

FIG. 4 shows the apparatus 100 (of FIGS. 1 and 2) during a tissuecollecting operation. During tissue collection, adipose tissue from alipoplasty procedure, which may be referred to as lipoasperate, isreceived in the tissue retention volume 112 or 148. FIG. 4 showsmaterial 224 that separate from lipoasperate, pass through the filter102 and collect in the filtrate volume 113, while tissue 220 (mostlyadipose tissue with some containments) is retained in the respectivetissue retention volume 112. The material 224 collecting in the filtratevolume 113 may be continuously or intermittently removed from thefiltrate volume 113 by suction through the suction port 118 and theconduit 120.

FIGS. 5 and 6 show the apparatus 100 of FIGS. 1 and 2 suspended by thehandle 128 following optional centrifuging of a washing operation andprior to digesting with a hypodermic needle 250 inserted through asealing mechanism 252 in the extraction port 110 for removal ofinfranatant (e.g., dirty wash liquid) from the filtrate volume 113 andinto the barrel of a syringe 254.

FIG. 7 shows the apparatus 100 of Design A following optionalcentrifuging of a washing operation and prior to digestion, in whichinfranatant (e.g., dirty wash liquid) is being removed from the filtratevolume 113 via a suction tube 260 inserted through the suction port 118and the conduit 120 into the filtrate volume 113, with the materialremoved from the filtrate volume 113 being collected within the barrelof a syringe 262.

FIG. 8 shows the apparatus 100 (FIGS. 1 and 2) being mounted on awarmer-shaker 270 for controlled temperature agitation of the contentsof the apparatus 100, such as may be performed during a digestionoperation after adding an enzyme to the internal containment volume. Themixers 124 (FIGS. 1 and 2) within the internal containment volume mayoptionally be operated during the shaking operation to assist agitationin addition to the shaking provided by the warmer-shaker 270. In anyevent, contents of the apparatus 100 may be agitated by the shakingmotion of the warmer-shaker

FIG. 9 shows two of the apparatus 100 (of FIGS. 1 and 2) received in acentrifuge 244 for centrifuging, as may be optionally performed during awashing operation or as may be performed during the centrifugingfollowing digestion.

FIG. 10 shows a tissue collection and processing apparatus 140 suspendedfrom the handle 168 and with a concentrate product being selectivelyremoved from collection chamber 154 through a sealing mechanism in anextraction port 156 through a hypodermic needle 288 into the barrel of asyringe 290. For example, the concentrate product being selectivelyremoved as shown in FIG. 10 may be a stromal vascular fraction fromadipose tissue.

FIG. 11 shows an apparatus 300 for collection of tissue comprisingadipose removed from a patient during a lipoplasty procedure and forpost-collection processing of collected tissue. The apparatus 300 isillustrated in a collection orientation. The collection orientation isthe orientation in which the apparatus 300 may be placed during thecollection of adipose removed from a patient during a lipoplastyprocedure. The apparatus 300 may also be placed in the collectionorientation during stages of the post-collection processing of collectedtissue as described below. Accordingly, subsequent references herein tothe orientation of the apparatus 300, such as top, bottom, lower andupper, will refer to the collection orientation of FIG. 11. Asillustrated, the apparatus 300 has an apparatus height H_(A), anapparatus length L, and an apparatus depth (or width) D. The apparatus300 also includes a suction port 302 and an inlet port 304. The suctionport 302 and inlet port 304 are disposed on the top of the apparatus 300when the apparatus 300 is in the collection orientation as illustratedin FIG. 11. In FIG. 11, and in certain other subsequent figures, theports are illustrated as having caps 328 thereon. Such caps 328 are usedto cover the various ports and may be removed and replaced as necessaryduring use of the apparatus 300.

The apparatus 300 includes a shell 306 and a lid 308. The shell 306 is aunitary bowl-like member where the only access into the interior, orcavity of the shell 306 is through the opening at the top of the shell306. As illustrated in FIG. 11, this opening at the top of the shell 306may be covered by the lid 308. The lid 308 and shell 306 may be rigid.The lid 308 and shell 306 are each preferably made of a clear polymericmaterial, such as a clarified polypropylene polymer composition, whichprovides low cellular adhesion and reasonable clarity. The lid 308 andshell 306 may be fabricated by injection molding. The lid 308 may beattached to the shell 306 in any appropriate manner, including snapping,clamping and/or gluing onto the shell 306. Together, the shell 306 andlid 308 form a container 322 with an internal containment volume 330(see FIG. 15 and accompanying discussion below) within the apparatus300. The internal containment volume 330 is the volume within the cavityof the shell 306 covered by the lid 308, and is the volume available fordisposing both hardware and material to be processed in the container322. This container 322 may have a container height H_(C). The shell 306may include a set of integral base supports 310 that may support theapparatus 300 in the collection orientation when the apparatus 300 isplaced on a horizontal surface. The apparatus height H_(A) is largerthan the container height H_(C) by the distance of projections above thetop of the container 322 for the inlet port 304, suction port 302, caps328 and other upward projecting features described below. The shell 306may be conveniently designed to efficiently fit within a centrifugebucket. The projections above the container height H_(C) may beconfigured so as not to interfere with operation of such a centrifuge.As seen in FIG. 11, the apparatus length L is equal to the containerlength and the apparatus depth is equal to the container depth (orwidth). As will be appreciated, the corresponding height, length anddepth dimensions of the internal containment volume 330 will equal theheight, length and depth dimensions of the container 322 less thecorresponding thicknesses of walls of the shell 306 and lid 308. Asshown in FIG. 11, some features may be integrally formed with the lid308. For example as shown in FIG. 11 the suction port 302 and the inletport 304 are integrally formed as a unitary fabricated piece with thelid 308. It should be appreciated that such features may be provided asseparate pieces and then assembled, such as by gluing or other means.For structural integrity, fabrication as a unitary piece is generallypreferred.

FIG. 12 shows another perspective view of the apparatus 300 with thecaps 328 to ports removed and with an installed handle 382. FIG. 13shows the apparatus 300 in the same orientation as in FIG. 12 with theshell 306 and handle 382 removed. With the shell 306 removed, a filter312 can be seen that is disposed within the internal containment volume330. The filter 312 may have a separation size in a range, for example,from 70 microns to 800 microns. The filter is preferably made of a meshmaterial. The preferred mesh material is a nylon mesh. Also visiblewithin the internal containment volume 330 is a suction port conduit 314extending downward from the suction port 302. Additionally, asillustrated in FIG. 13, all components of the apparatus 300, except forthe shell 306, are interconnected to the lid 308. In this regard, thesubassembly shown in FIG. 13 may be assembled as shown and inserted intothe shell 306.

FIG. 14 shows another perspective view of a portion of the apparatus 300in the same orientation as in FIG. 13 with both the shell 306 and thefilter 312 removed. With the filter 312 removed, a flow barrier skirt324 extending downward from the lid 308 into the internal containmentvolume 330 is visible. In an example, the flow barrier skirt 324 mayextend between 5 mm and 50 mm downward from the lid 308. The flowbarrier skirt 324 may serve as an attachment point for the filter 308such that the filter 312 may be fixed relative to the lid 308. The flowbarrier skirt 324 may also serve to prevent material from entering atissue retention volume 332 (described below) and immediately movingthrough the filter 312 into the filtrate volume 334. The tissueretention volume 332 is that portion of the internal containment volume330 contained within the filter 312 and barrier skirt 324 below the lid308. The filtrate volume 334 is that portion of the internal containmentvolume 330 disposed outside of the filter 312 and barrier skirt 324.With the flow barrier skirt 324 in place, and material entering theinlet port 304 must at least move to below the lowest level of the flowbarrier skirt 324 before it is able to pass through the filter 312 intothe filtrate volume 334. The flow barrier skirt 324 may be part of afilter subassembly that includes the flow barrier skirt 324 and thefilter 312. This subassembly is mounted to the lid 308 with four screws326.

The filter 312 is asymmetric with respect to the lid 308 and shell 306in that it is configured to provide clearance between its left side (asviewed in FIG. 13) and the shell 306 for the suction port 302 andsuction port conduit 314. A portion of the filter 312 may be disposedabout (e.g., rest on or around) a portion of the suction port conduit314.

With the filter 312 removed (FIG. 14), a mixing device 316 can be seen.The mixing device 316 includes a rotatable shaft 318 and a set of mixingmembers 320. The axis of rotation of the rotatable shaft 318 may bethrough a central axis of the rotatable shaft 318. The mixing members320 are in the form of paddles extending outward from the rotatableshaft 318. Accordingly, when the rotatable shaft 318 is rotated, themixing members 320 will be rotated through the materials within thetissue retention volume 332 to aid in mixing the materials within theinternal containment volume 330, and in particular within the tissueretention volume 332. The rotatable shaft 318 extends from outside ofthe internal containment volume 330 through the lid 308 to the inside ofthe internal containment volume 330. As the rotatable shaft 318 isrotatable relative to the lid 308, the mixing members 320 fixed to therotatable shaft 318 are also rotatable relative to the lid 308. Therotatable shaft 318 may be made from a metal composition, such asstainless steel (e.g., grade 303, 304, or 316). Alternatively, therotatable shaft 318 may be made from a high-strength polymer compositionsuch as an Ultem™ resin product.

The rotatable shaft 318 may include a handle interface 380 (FIG. 13)that may provide an interface for the handle 382 (FIG. 12) to beinterconnected to the portion of the rotatable shaft 318 outside of theinternal containment volume 330. The handle interface 380 of FIG. 13 isin the form of a pair of parallel surfaces disposed about the portion ofthe rotatable shaft 318 outside of the internal containment volume 330.The handle 382 has a mating pair of interior parallel surfacesconfigured such that when the handle 382 is placed over the handleinterface 380, turning the handle 382 will result in turning therotatable shaft 318 and the mixing device 316. Such an interface 380also allows for the handle 382 to be removed from and replaced on thehandle interface 380 as needed during use of the apparatus 300.

FIG. 15 is a side schematic view of the apparatus 300 showing the mixingdevice 316 and filter 312 within the shell 306. The internal containmentvolume 330 is the entire volume within the shell 306 and under the lid308. Together, the portions of the shell 306 and lid 308 that containthe internal containment volume 330 are a container 322 of the apparatus300. The filter 312 divides and separates the internal containmentvolume 330 of the container 322 into the tissue retention volume 332disposed inside the filter 312, and a filtrate volume 334 disposedwithin the shell 306 on the outside of the filter 312. The filtratevolume 334 is that portion of the internal containment volume 330 intowhich filtrate enters after passing through the filter 312 from thetissue retention volume 332.

Disposed within the internal containment volume 330 at the bottom of theshell 306, below a level 350 that is at or below the lowest extent ofthe filter 312 (and therefore also below the lowest extent of the tissueretention volume 332), is a collection volume 336, such that thecollection volume 336 is part of the filtrate volume 334 and occupiesthe lowermost portion of the filtrate volume 334 located below thelowest elevation of the tissue retention volume 332.

The shell 306 has a tapered wall portion 338 that defines a taperedportion 340 of the internal containment volume 330, such that thecross-sectional area of the tapered portion 340 of the internalcontainment volume 330 tapers with a reducing cross-sectional area in adirection toward bottom of the container 322. By tapering, it means thatthe cross-sectional area in a horizontal plane (assuming the apparatus300 is in the collection orientation) becomes smaller in the directionof the taper (e.g., a direction orthogonal to the horizontal plane). Thetapered portion 340 of the internal containment volume 330 occupies theportion of the internal containment volume 330 below a level 352 wherethe tapered wall portion 338 meets a straight wall portion 342 of theshell 306. The tapered wall portion 338 is shown as having a flat,uniform inclined wall surface. The incline angle of surfaces of thetapered wall portion need not be uniform from the top to the bottom ofthe tapered portion 340 of the internal containment volume 330, and mayvary from top to bottom with portions having different incline angles,and may have a curved surface, provided that the cross-section area isreducing in the direction of the taper. Also, the tapered wall portion338 need not be uniform around the perimeter of the tapered portion 340of the internal containment volume 330. For example, in the embodimentin FIGS. 13-15, the tapered wall portion 338 has a steeper incline onthe ends than on the front or back of the apparatus 300.

The shell 306 may comprise an upper portion 344 generally above a level354 and having a first wall surface portion 348 defining a correspondingupper portion 368 of the internal containment volume 330. Substantiallyall of the first wall surface portion 348 may have an incline relativeto horizontal of at least 75°. For example, substantially all of thefirst wall surface portion 348 may be substantially vertical (90°incline relative to horizontal). The shell 306 may include a lowerportion 346 located below the upper portion 344 and having a second wallsurface portion 358 defining a corresponding lower portion 360 of theinternal containment volume 330. The lower portion 360 may include thetapered wall portion 338 defining the tapered portion 340 of theinternal containment volume 330. Substantially all of the tapered wallportion 338 may preferably have an incline relative to horizontal in arange of from 30° to 60°, although other angles or curved surfaces maybe used. The tapered portion 340 of the internal containment volume 330may occupy substantially the entire lower portion 360 of the internalcontainment volume 330. At least a first portion 362 of the filter 312may be disposed in the upper portion 368 of the internal containmentvolume 330 and a second portion 364 of the filter 312 may be disposed inthe lower portion 360 of the internal containment volume 330. Thetapered wall portion 338 may form a nadir 384 at its lowest elevation.The nadir 384 may also be a nadir of the collection volume 336, thefiltrate volume 334, the container 322, and the internal containmentvolume 330.

The internal containment volume 330 may include an available processingvolume or “useable” volume 366 which may be the portion of the internalcontainment volume 330 that is usable and/or may normally be occupied bymaterials within the container 322 during normal use. For example, theavailable processing volume 366 may be the portion of the internalcontainment volume 330 below a level 356 that coincides with the bottomextension of a port through the lid 308 (such as a second suction port370 discussed below, see FIG. 11) and that is not occupied by portions(e.g., internal hardware) of the apparatus 300 within the internalcontainment volume 330, such as the mixing device 316, barrier skirt324, filter 312 and suction port conduit 314. The top of the availableprocessing volume may be at the elevation of the bottom extension of theinlet port 304, which may define a maximum fill level within theinternal containment volume 330.

The inlet port 304 in fluid communication with the tissue retentionvolume 332 through the lid 308 is configured for introducing tissuecomprising adipose directly into the tissue retention volume 332 duringa lipoplasty procedure. However, use of the apparatus 300 is not solimited, and the tissue may be introduced into the apparatus usingtissue previously collected in another container and transferred to theapparatus 300. An additional access port 372 in fluid communicationthrough the lid 308 with the tissue retention volume 332 provides anadditional route into the tissue retention volume 332, for example forintroduction of additives.

The suction port 302 is in fluid communication through the lid 308 withthe filtrate volume 334 via suction port conduit 314 extending from thesuction port 302 to within the tapered portion 340 of the internalcontainment volume 330 in the vicinity of the top of the collectionvolume 336. The suction port 302 is configured for connection to avacuum system, for example through connection of a suction conduitthrough which suction may be applied by a vacuum system to suction fromthe filtrate volume 334 material passing through the filter 312 from thetissue retention volume 332 into the filtrate volume 334.

Referring now to FIGS. 15, 28 and 29, the rotatable shaft 318 mayinclude a filter contact member 376 that is offset from an axis ofrotation of the rotatable shaft 318. A lower end of the filter contactmember 376 may contact a portion of the filter 312 as illustrated inFIG. 15. As the rotatable shaft 318 is rotated, the filter contactmember 376 may rotate in a circular path about the axis of rotation ofthe rotatable shaft 318 remaining in contact with and moving along aportion of the filter 312. This contact may cause the filter 312 todeform and such deformation and/or the contact between the filtercontact member 376 and filter 312 may cause materials that may haveadhered to the filter 312 in this region to become dislodged from thefilter 312. Thus, the filter contact member 376 may assist in keepingthe filter from clogging and increasing the effectiveness of the filter312.

The rotatable shaft 318 may include a lumen 374 therethrough. The top ofthe lumen 374 is visible in FIG. 13 and the bottom of the lumen 374 isat the opposite end of the rotatable shaft (FIG. 14). The bottom of thelumen 374 is visible in FIG. 29. The lumen 374 may have a distal end 386(FIG. 14, 29) within the tissue retention volume 332 and a proximal end388 (FIGS. 13 and 14) outside of the internal containment volume 330 andthus may allow access to the tissue retention volume 332 therethrough.The lumen 374 may be disposed along the central axis of the rotatableshaft 318. The lumen 374 thus provides a conduit for accessing theinternal containment volume 330. As further described below, the lumen374 may provide access for removing processed material from the internalcontainment volume 330. In that respect, the opening through the lid 308through which the rotatable shaft 308 extends acts as an extraction portthrough which access is provided via the lumen 374 that passes throughsuch opening. The apparatus 300 may include a plug 378, shown in FIG. 14and not shown in FIG. 13, that may be placed in the proximal end 388 oflumen 374 to seal the lumen 374.

As illustrated in FIGS. 28 and 29, the apparatus 300 may include anoptional barrier member 390 (shown in FIG. 28, absent from FIG. 29). Thebarrier member 390 may be secured to the mixing device 316 via twoscrews 391 or by any other appropriate means such as snaps or by beingmolded integrally with the mixing members 320.

As shown in FIG. 16, in configurations where the barrier member 390 isnot present, a hypodermic needle 392 may be inserted through the lumen374 and may be advanced out of the distal end 386 of the lumen 374 andto pierce through the filter 312 to directly access the collectionvolume 336 (the volume under the line 410 in FIG. 16). Thus, without thebarrier member 390 present, the hypodermic needle 392 may be used toinject material into, or remove material from the collection volume 336.Additionally, as the axis of the lumen 374 is vertically oriented,access to the collection volume 336 using the hypodermic needle 392 isby downward vertical insertion into the lumen 374 from above thecontainer. Such vertical insertion coupled with the ability of theapparatus 300 to be placed on a flat surface in the collectionorientation, allows for user-friendly access to the collection volume336, and helps avoid complications that could compromise operations tocollect valuable processed material from the collection volume 336.

The hypodermic needle 392 may be interconnected to a syringe 394. Theproximal end 388 of the lumen 374 may include a tapered receptacleadapted to mate with a tapered tip of the syringe 394. In this regard,as shown in FIG. 16, the depth of penetration by the hypodermic needle392 into the collection volume 336 when the tapered tip of the syringe394 is in contact with the tapered receptacle of the lumen 374 may becontrolled by controlling the length of the hypodermic needle 392extending from the syringe 394. Additionally, the proximal end 388 ofthe lumen 374 may include a feature, such as a notch, to retain ano-ring (not shown) such that when the syringe 394 is positioned againstthe proximal end 388 of the lumen 374, the o-ring forms a seal betweenthe proximal end 388 of the lumen 374 and the syringe 394 (i.e., a sealthrough the o-ring between a wall surface in the tapered receptacle andan exterior wall surface of the tip of the syringe inserted into thetapered receptacle).

In configurations where the barrier member 390 is present, as shown inFIG. 28, direct access from the lumen 374 to the collection volume 336is prevented. Furthermore, the distance between the barrier member 390and the distal end 386 of the lumen 374 may be selected to achieve adesired flow restriction through a gap between the distal end 386 of thelumen 374 and the barrier member 390. For example, the distance betweenthe barrier member 390 and the distal end 386 of the lumen 374 may bebetween one and five millimeters. Such a distance may be beneficial whenthe apparatus 300 is employed to perform a fat graft and the lumen 374is used to remove tissue from the tissue retention volume 332. Bymaintaining an appropriate standoff between the barrier member 390 andthe distal end 386 of the lumen 374, and by configuring the barriermember with an appropriate areal extension beyond the perimeter of theopening of the distal end 386 of the lumen 374 (e.g., the barrier member390 is a large enough plate), potential for flow short-circuiting todraw in air or other fluid through the filter 312 from the filtratevolume 334 may be significantly reduced or avoided when processedmaterial (e.g., for a fat graft) is extracted by suction through thelumen 374.

Referring to FIGS. 17A and 17B, the second suction port 370 includes atranslatable member 396 that may be translated up and down relative tothe lid 308 to vary the depth (elevation within the filtrate volume 334)at which material from the filtrate volume 334 is drawn through thesecond suction port 370. Examples of the various depths (elevations) atwhich the translatable member 396 may be positioned are illustrated inFIGS. 17A, 19, 20 and 22 and are discussed below in relation to methodsof using the apparatus 300. The fit between the translatable member 396and the opening through the lid 308 of the second suction port 370 issuch that the translatable member 396 may be readily translated up anddown to a desired level, while maintaining a tight enough fit to allow avacuum applied to the translatable member 396 to adequately drawmaterial out of the filtrate volume 334.

As illustrated in FIG. 18, the apparatus 300 may include a pre-filterunit 400 fluidly connected to the inlet port 304 via a length of tubing402. The pre-filter unit 400 may comprise within a housing a pre-filter,which may be in the form of a mesh screen with openings, for example,preferably in a range of from 0.5 millimeter to 2 millimeters. Thepre-filter unit 400 may be used to pre-filter tissue prior tointroduction into the tissue retention volume 332 of the apparatus 300.The tissue being pre-filtered may be supplied from a lipoplasty cannulaused during a lipoplasty procedure. Moreover, the suction port 302 ofthe apparatus 300 may be fluidly connected with a canister 206 via asuction conduit 208 similar to as illustrated in FIG. 3.

In general, the parts discussed with reference to the apparatus 300 maybe made from any appropriate biocompatible material. In particular, theshell 306 may be made from a biocompatible transparent polymer materialto allow inspection of the contents therein. Screws 326 and therotatable shaft 318 may be made from metal, such as stainless steel.Other parts of the assembly 300 pictured in FIG. 11 may be made fromappropriate biocompatible polymers.

Various exemplary dimensions of one specific nonlimiting example of anapparatus 300 will now be described with reference to FIGS. 11 and 15.In this example, the apparatus 300 has apparatus height H_(A) of about157 mm, an apparatus length L of about 145 millimeters, and an apparatusdepth D of about 126 millimeters. The containment volume height H_(C) isabout 124 millimeters. The example has an available processing volume366 of about 760 milliliters and a collection volume 336 of about 23milliliters. The portion of the tissue retention volume 332 thatcoincides with the available processing volume 366 is about 580milliliters. As will be appreciated, a milliliter is equal in volume toa cubic centimeter, and the volumes listed here in milliliters may beequivalently stated as cubic centimeters.

In a method for processing tissue from a lipoplasty procedure using theapparatus 300, the tissue is processed within the internal containmentvolume 330 to prepare within the apparatus 300 a concentrated productcomprising at least one target component, or at least one targetmaterial, from the tissue. Many features of the previously discussedmethods may also be employed in the current method where appropriate.Such features include, inter alia, multiple washings, shaking, heating,and centrifuging as previously described. Returning to the presentmethod, the tissue is introduced into the tissue retention volume 332through the inlet port 304. The tissue may be pre-filtered usingpre-filter unit 400 prior to being introduced into the tissue retentionvolume 332. The method may comprise washing tissue in the internalcontainment volume 330 with a wash liquid. Optionally, the washing mayinclude centrifuging the apparatus 300. After washing, the method maycomprise digesting tissue within the internal containment volume 330.After the digestion, the method may include centrifuging the apparatus300 to prepare in the collection volume 336 a concentrate productcomprising at least one target component. For example the concentrateproduct may comprise, or may consist essentially of, stromal vascularfraction from adipose tissue, and a target component may be stem cellsfrom adipose tissue.

During the washing, the wash liquid may be added to the internalcontainment volume 330 to contact tissue within the tissue retentionvolume 332 and with at least a portion, preferably a majority, and morepreferably most, of the wash liquid passing through the filter 312 intothe filtrate volume 334. The addition of the tissue to the internalcontainment volume 330 may occur simultaneously with the wash liquidbeing removed from the filtrate volume 334 via vacuum applied to thesuction port 302. In this regard, a volume of tissue larger than theinternal containment volume 330 may be introduced into the internalcontainment volume 330 during the performance of the method. Moreover,the removal of wash liquid may continue after the introduction of tissueinto the internal containment volume 330 has stopped.

The wash liquid may wash one or more components from the tissue whileretaining washed tissue in the tissue retention volume 332. The washedtissue may be retained in the tissue retention volume 332 by the filter312. Wash liquid passing into the filtrate volume 334 may be removedfrom the filtrate volume 334, along with any component or componentswashed from the tissue. Optionally, after adding the wash liquid, theapparatus 300 may be centrifuged to facilitate a high degree ofseparation of the wash liquid from the tissue retained in the tissueretention volume 332. Next, the wash liquid may be removed from thefiltrate volume 334 by suctioning through the suction port 302 of theapparatus 300. The washing may include multiple wash stages. During thewashing, the mixing device 316 may be rotated by rotating the handle 382to mix contents of the internal containment volume and assist thewashing process.

During the digestion, an enzyme, such as for example collagenase, may beadded to the internal containment volume 330 through the additionalaccess port 372 or through the inlet port 304. During the digesting, themixing device 316 may be rotated to assist in the digesting process.

After adding the enzyme, the digesting may comprise agitating contentsof the containment volume of the apparatus 300 for a time and at atemperature sufficient for the digestion to proceed to an extent tosignificantly release the target component, or material, in the desiredform capable of passing through the filter 312. The agitating mayinvolve any method to agitate contents of the internal containmentvolume 330, including for example one or both of: (a) shaking theapparatus 300 to agitate the contents within the apparatus 300 and (b)mixing the contents within the apparatus 300 by rotating the mixingdevice 316 using the handle 382.

Post-digestion centrifuging promotes separation of the target componentfrom the digested tissue and passage of the target component through thefilter 312 for collection in the collection volume 336. The targetcomponent may include leuko stromal vascular cells (e.g., stem cells)from adipose tissue. As illustrated in FIG. 19, multiple material phasesmay collect within the filtrate volume 334 as a result of thecentrifuging. The first (bottom) material phase may be a small layer ofred blood cells 428 located in the region of the filtrate volume 334below the line 408. This volume below the line 408 occupies a bottomportion of the collection volume 336, and in many situations may evennot be present or may be so small as to be indistinguishable. The secondmaterial phase may be a stromal vascular fraction layer 430 from adiposetissue and may be located in the region of the filtrate volume 334 belowthe line 412 and above the line 408. As will be appreciated, the redblood cell layer 428 and the stromal vascular fraction layer 430 may notbe divided by a sharp line, and the blood cell layer 428 may grade intothe lower portion of the stromal vascular fraction layer 430. Thisvolume below the line 412 and above the line 408 also occupies a portionof the collection volume 336. The stromal vascular fraction layer 430,or the stromal vascular fraction layer 430 together with the red bloodcell layer 428, may be in the form of a pellet. A third material phasemay be an aqueous layer 432 that occupies the region of the filtratevolume 334 below the line 406 and above the line 412. A fourth materialphase may be a disaggregated adipose layer 434 that occupies the regionof the filtrate volume 334 below the line 414 and above the line 406. Afifth material phase may be an oil layer 436 that occupies the region ofthe filtrate volume 334 below the line 416 and above the line 414. Theseparated phase layers as shown are provided to illustrate relativepositioning and are not intended to represent an actual scale of therelative sizes of the phases, except that the red blood cell layer 428and stromal vascular fraction layer 430 are contained within thecollection volume 336 and the other layers extend above the collectionvolume. As will be appreciated, the material phases 428, 430, 432, 434and 436 are in order of decreasing density, with red blood cell layer428 being the most dense phase and with the aqueous layer 432, thedisaggregated a dispose layer 434 and the oil layer 436 all being lessdense than the stromal vascular fraction layer 430.

The translatable member 396 of the second suction port 370 may beemployed to first remove the oil layer 436, then to remove thedisaggregated adipose layer 434, and then to remove the aqueous layer432. As illustrated in FIG. 19, the translatable member 396 may bepositioned such that the end of the translatable member 396 is disposedwithin the oil layer 436. Suction applied to the translatable member 396will remove the As fluid is removed, the translatable member may belowered to remove additional fluid down to a desired level, which may beremoval of all or most of layers 436,434 and 432. For example, once theoil layer 436 has been removed, the translatable member 396 may belowered into the disaggregated adipose layer 434 and then the aqueouslayer 432 for sequential removal of these layers. FIG. 20 illustratesthe aqueous layer 432 partially removed (after already removing the toplayers 436 and 434 such that the top of the aqueous layer 432 is at line418). As another example, the translatable member 396 may be initiallyinserted to the position shown in FIG. 20 and suction applied until aportion of the aqueous layer 432 is removed and also the disaggregatedadipose layer 434 and oil layer 436 are removed above line 418,resulting in the arrangement of FIG. 20.

Once fully inserted into the filtrate volume 334, the translatablemember 396 may not be operable to remove a portion of the aqueous layer432 while the apparatus 300 is in the collection orientation.Accordingly, a user may gently tilt the apparatus 300 as illustrated inFIG. 21 to further remove the aqueous layer 432. As illustrated, thestromal vascular fraction 430 below the line 412 may form a pellet whichmay retain its position as the apparatus 300 is tilted. This attributeof the pellet allows the apparatus 300 to be tilted such that theaqueous layer 432 flows laterally toward the translatable member 396disposed proximate to the interface between the tapered wall portion 338of the shell 306 and the straight wall portion 342 of the shell 306 asillustrated by line 420 in FIG. 21. Such tilting can allow suction to beapplied to the aqueous layer 432 without the suction substantiallyaffecting the stromal vascular fraction 430, which remains in place andstationary relation to the container. Once the aqueous layer 432 hasbeen satisfactorily removed, the apparatus 300 may be returned to itscollection orientation, as shown in FIG. 22, for removal of the stromalvascular fraction layer 430 from the collection volume 336.

Next, the hypodermic needle 392 may be inserted into the collectionvolume 336 as illustrated in FIG. 16 and a diluent fluid (e.g.,suspension liquid) may be injected into the collection volume 336 suchthat the diluent fluid, stromal vascular fraction layer 430 and thelayer of red blood cells 428 together occupy at least a portion of thecollection volume 336 under line 410, and are preferably limited tobeing present only in the collection volume 336 and do not occupy spaceabove line 410. After injection of the diluent fluid, a user may gentlytap the apparatus 300 against a hard surface to cause the diluent fluidto mix with the stromal vascular fraction and the layer of red bloodcells. A second hypodermic needle may then be inserted through the lumen374 and the diluent/stromal vascular fraction/red blood cell mixture maybe removed from the apparatus 300, for example by drawing the mixturethrough the hypodermic needle and into a syringe.

Referring now to FIGS. 23 and 24, a further embodiment is shown for atissue collection and processing apparatus. As shown in FIGS. 23 and 24,a tissue collection and processing apparatus 450 has a collectionorientation in a freestanding, upright position as supported by basesupports 452. The apparatus 450 includes a lid 454 covering a bowl-likeshell 456, which make up a container having an internal containmentvolume under the lid 454 within the shell 456. The apparatus 450includes a first suction port 458, a second suction port 460, an inletport 462 and an auxiliary access port 464, which may be generally asdescribes for similar features of the apparatus 300 described withrespect to FIGS. 11-17.

The first suction port 458 is connected with a suction conduit 466extending from the first suction port 458 to within a tapered portion ofan internal containment volume of the apparatus 450. The second suctionport 460 is adapted to receive a translatable suction conduit, similarto the translatable member 396 described with respect to FIG. 17. Theapparatus 450 includes a filter 468 suspended from the lid 454 and whichdivides the internal containment volume in the apparatus between atissue retention volume disposed inside the filter 468 and a filtratevolume disposed on the other side of the filter 468. The apparatus 450includes a rotatable mixer disposed within the filtrate volume thatincludes propellers 470 connected to a rotatable shaft 472, which may berotated to operate the rotatable mixer and cause the impellers 470 tomix and circulate fluid within the internal containment volume of theapparatus 450. The propellers 470 may alternatively be referred to asimpellers or mixing impellers. The rotatable shaft 472 includes aninternal lumen that extends from a proximal end outside of the containerof the apparatus to a distal end in the tissue retention volume, topermit access into the internal containment volume in a manner similarto that discussed previously for the apparatus 300 shown in FIGS. 11-17.A removable plug 474 may be disposed in a proximal end of the lumen forsealing the lumen when the lumen is not in use. The rotatable shaftincludes a handle interface which may be interfaced with ahand-manipulable handle 476 (FIG. 24) to operate the rotatable mixer.The rotatable mixer is designed for primary operation by rotating thehandle in a clockwise direction, as indicated by the directional arrowson a plate as shown in FIG. 14. The apparatus 450 includes attached caps478 which may be used to cap the first suction portion 458, secondsuction port 460 and inlet port 462 as needed, such as to seal thecontainer for transportation between processing locations or duringagitation on a warmer-shaker during digestion operations. The apparatus450 is operable substantially in the same way as described previouslyfor the apparatus 300 shown in FIGS. 11-17. The apparatus 450 includesvolume gradation markings 480 that indicate the volume contained withinthe tissue retention volume (within the filter 468) up to differentelevations of the container 450 when in the access orientation.

With continued reference to FIGS. 23 and 24 features of one or both ofthe propellers 470 may be configured to assist mixing of contents withinthe portable container apparatus and to reduce potential for plugging ofthe filter 468. One or both of the propellers 470 may have pitchedblades that direct flow of fluid from the respective propeller 470 in anaxial direction relative to the axis of rotation of the rotatable shaft472. As shown in FIG. 24, the configuration of the bottom propeller 470may include impeller blades 494 that are pitched at an angle that willpropel fluid flow in an upper axial direction along the rotatable shaft472 when the rotatable shaft 472 is rotated in the clockwise rotationaldirection. This type of upward pumping action by the bottom propeller470 may assist in moving material from the filter 468 to help keep thefilter 468 from plugging. In similar manner, the top propeller 470 mayhave pitched blades 493 that propel fluid flow in an axial directionupward toward the underside of the lid 454 and away from the tissuecollector 482 when the rotatable shaft 472 is rotated in the clockwiserotational direction. This upward pumping action by the top propeller470 may assist in further pulling material up and away from the filter468 to help prevent plugging of the filter 468.

In one enhancement, one or more of the blades 494 may be configured toscrape at least a portion of the filter 468 when the rotatable shaft472, and thus also the bottom propeller 470, is rotated in the clockwiserotational direction. Such scraping of the filter 468 may beaccomplished by configuring an edge portion 495 of a blade 494 tocontact and scrape surfaces of the filter 468. In that regard, a blademay be configured with a slanted lower edge shaped to correspond withand contact a corresponding tapered portion of the filter 468. A leadingedge of the blade 494 may have a tapering width to assist in scrapingtissue or other material away from the surface of the filter 468. Forexample, the configuration of the blade 494 may include a beveledsurface toward a leading edge of a slanted edge portion that contactsthe filter 468 and that may help to lift tissue or other material awayfrom the filter 468 when the lower propeller 470 is rotated in theclockwise rotational direction.

FIG. 25 as a generalized process block diagram illustrating oneembodiment of a method of the invention. As shown in FIG. 25, the methodincludes a washing step 502, during which adipose tissue disposed withina portable container is washed to remove contaminants from the adiposetissue. Contaminants that may be associated with the adipose tissueinclude for example blood, free lipids, small particles and debris andother materials that may have been collected with the adipose tissue orresult from degradation during a tissue collection operations.

The washing 502 may include one or, preferably, multiple wash cyclesduring which adipose tissue is washed with wash liquid within thecontainer. The wash liquid, for example, may be a buffer solution, suchas Lactated Ringer's solution or Hank's Balanced Solution, and may haveadditional additives, such as one or more of an anti-clotting agent, anantibiotic and an antifungal. An anti-clotting agent may beneficiallyprevent coagulation of blood that may be present, and may assisteffective washing of blood from the adipose tissue. Antibiotics andantifungals may help protect against problems associated withinadvertent outside contamination of the adipose tissue within thecontainer. Such a wash liquid may also include one or more additionalbuffering agents, such as glycine. One preferred material for use as ananti-clotting agent is heparin.

During a wash cycle, the wash liquid is mixed with the adipose tissue inthe container and then preferably substantially all of the wash liquidwith washed contaminants from the adipose tissue is removed from thecontainer from a first side (filtrate volume side) of a filter withinthe container while retaining the washed adipose tissue in the containeron a second side (tissue retention volume side) of the filter.

The washing may include any of the feature refinements and additionalfeatures discussed above.

After the washing 502, the washed adipose tissue in the container issubjected to a digesting step 504. Digestion medium comprising acollagenase enzyme solution is added to the container to contact thewashed adipose tissue. The digestion medium is added in a volume ratioof from 0.6:1 to 2:1 of digestion medium: adipose tissue. The digestionmedium contains collagenase enzyme in an amount to provide from 150 to300 collagen digestion units (CDU) per milliliter of catalytic volume.Catalytic volume refers to the total volume of the digestion medium andadipose tissue within the container to which the digestion medium isadded. After the digestion medium is added to the container, enzymaticdigestion within the container is permitted to proceed for a retentiontime of from 20 minutes to 50 minutes while the container is disposed ina temperature controlled environment maintained within a temperaturerange of from 32° C. to 38° C., and with at least occasional, andpreferably substantially continuous, agitation of contents to thecontainer. The digesting step 504 may include any of the featurerefinements and additional features discussed above.

The method as shown in FIG. 25 also includes a stopping digestion step506 occurring after the digesting step 504. The stopping digestion stepshould occur no earlier than the end of the retention time for theenzymatic digestion in the temperature controlled environment, but inany event should be performed within 50 minutes following adding thedigestion medium to the container during the digesting step 504. Thestopping digestion step 506 includes adding a stopping reagent to thecontainer to positively stop enzymatic activity within the container.This is important, because if enzymatic activity is not discontinued,digestion within the container may proceed to an undesirable degree inwhich the enzyme may destroy the viability of a significant number ofthe leuko stromal vascular cells.

As shown in FIG. 25, the method includes, after the stopping digestionstep 506, a centrifuging step 508. The centrifuging step 508 isperformed with the container disposed in a centrifuge and the centrifugeis operated to centrifuge the container to form density-separated phaseswithin the container. These density-separated phases include ahigher-density pellet phase rich in leuko stromal vascular cells, whichpellet phase may form adjacent a bottom of the container. Thedensity-separated phases also include lower-density material phases. Bylower-density, it is meant that the lower-density material phases have alower-density than the pellet phase. When the container is oriented withthe pellet phase adjacent a bottom of the container (e.g., in a anaccess orientation for the container), the lower-density material phaseswill be disposed in the container above the pellet phase. Thelower-density material phases may include, in order of decreasingdensity, an aqueous layer, a disaggregated adipose layer (containingremnants of disaggregated adipose tissue) and an oil layer. The pelletphase is enriched in, and may be mostly or even substantially entirelycomprised of, leuko stromal vascular cells (e.g., stromal vascularfraction). On a side of the pellet phase opposite the lower-densitymaterial phases may be disposed a small red blood cell phase. Providedthat washing of the adipose tissue is thorough during the washing step502, this red blood cell phase may be extremely small, and in some casemay be difficult to distinguish from a bottom portion of the pelletphase. The centrifuging step 508 may include any of the featurerefinements and additional features discussed above.

After the centrifuging step 508 has been completed, the container isremoved from the centrifuge and subjected to a step 510 of removinglower-density phases. During the step 510, the lower-density materialphases are removed from the container while the pellet phase is retainedwithin the container, preferably while maintaining pellet phase in anundisturbed state, in place at the location of the container where thepellet collected during the centrifuging step 508. The step 510 mayinclude any of the feature refinements and additional features asdiscussed above.

As shown in FIG. 25, the stopping digestion step 506 is performed afterthe digesting step 504 and prior to the centrifuging step 508. Suchsequencing is preferred, but not required. In one variation, thestopping digestion step 506 may be performed after the centrifuging 508.However, because enzymatic digestion would continue during thecentrifuging, such a variation in the sequence is not preferred, toprovide better control over the timing and extent of the enzymaticdigestion.

The leuko stromal vascular cells, which include stem cells, contained inthe pellet phase represent valuable product. For effective use of thesevaluable leuko stromal vascular cells, it is generally necessary toremove the cells from the container. This has been a significant problemin the context of using multi-step portable containers for processingthat is addressable with various implementations of the invention.

Referring now to FIG. 26, another embodiment of implementation of amethod of the invention is shown. The implementation shown in FIG. 26includes the washing step 502, the digesting step 504, the stoppingdigestion step 506, the centrifuging step 508 and the removing oflower-density phases step 510 as discussed with FIG. 25. As shown inFIG. 26, after the removing lower-density phases step 510, the methodincludes a dispersing cells step 512. During the dispersing cells step512, aqueous suspension liquid is introduced into the container to mixwith the pellet phase and to act as a dispersion medium for dispersingcells of the pellet phase in the suspension liquid. Dispersion of cellsfrom the pellet phase may be aided by tapping the container to dislodgeand break up the pellet phase to assist effective dispersion of theleuko stromal vascular cells in the suspension liquid. The dispersingcells step 512 may include any of the feature refinements and additionalfeatures as discussed above.

After the dispersing cells step 512, the processing shown in FIG. 26includes a removing dispersed cells step 514, during which most, andpreferably substantially all, of the suspension liquid with thedispersed cells from the pellet phase is removed from the container,thereby recovering the leuko stromal vascular cells from the container.The removing dispersed cells step 514 may include any of the featurerefinements and additional features discussed above.

The method of the invention includes multi-step processing in a singleportable container, such as for example the container of a tissuecollection and processing apparatus as described above. The method isdesigned to address inherent problems with multi-step processing in asingle container. Through careful design of the container (e.g., asdescribed with tissue collection and processing apparatus describedabove) and combined processing specifics (e.g., washing, digesting,centrifuging, and cell removal specifics), processing in a portablecontainer may be performed efficiently and effectively to process largequantities of collected adipose tissue relative to the total volume ofthe container and to effectively recover a large number of viable leukostromal vascular cells released from the processed adipose tissue. Thisis remarkable given the inherent design deficiencies associated with theuse of a single container for performing multiple steps relative to anintegrated multi-container processing system with different containersdedicated to different steps of processing.

Reference is now made to FIG. 27, which illustrates portability of thecontainer during implementation of a method of the invention. As shownin FIG. 27, processing may be performed at a facility providing multipledistinct locations where the portable container may be temporarilylocated to perform different processing operations within the container.FIG. 27 shows three such possible distinct processing locations: a workstation, a centrifuge and a temperature controlled environment. As shownby the arrows in FIG. 27, the portable container may be transportedbetween these different locations to perform different process tasks oroperations, for example processing tasks and operations associated withthe method implementations of FIGS. 25 and 26. The transportation mayconveniently be accomplished by a person carrying the portable containerbetween the different locations.

The work station 520 may include a flat work surface, such as a flatsurface of a table, bench or counter where the portable container may bestably positioned in an access orientation for convenient access to addmaterial to or remove material from the portable container. In apreferred implementation, when the portable container is in such anaccess orientation, all access into the container for adding andremoving materials is in a downward direction from above the container.Operations that may be performed at such a work station 520 include, forexample, one or more of the washing step 502, the stopping digestionstep 506, the removing lower-density phases step 510, the dispersingcells step 512, and the removing dispersed cells step 514 shown in FIGS.25 and 26. Adding digesting medium to the container may also beconveniently performed at such a work station 520. As will beappreciated, an actual facility may include multiple work stations,rather than a single work station as shown in FIG. 27. For exampledifferent work stations may be specifically designed and equipped forperforming specific processing tasks. Such multiple work stations may becontiguous or at separated locations in a facility.

Enzymatic digestion within the container for a retention time may beperformed at the temperature controlled environment 522. The temperaturecontrolled environment may be provided by a warmer-shaker, for example.

Centrifuging (e.g., step 508 of FIGS. 25 and 26) may be performed at thelocation of the centrifuge 524 shown in FIG. 27.

The foregoing discussion of the invention and different aspects thereofhas been presented for purposes of illustration and description. Theforegoing is not intended to limit the invention to only the form orforms specifically disclosed herein. Consequently, variations andmodifications commensurate with the above teachings, and the skill orknowledge of the relevant art, are within the scope of the presentinvention. The embodiments described hereinabove are further intended toexplain best modes known for practicing the invention and to enableothers skilled in the art to utilize the invention in such, or other,embodiments and with various modifications required by the particularapplications or uses of the present invention. It is intended that theappended claims be construed to include alternative embodiments to theextent permitted by the prior art. Although the description of theinvention has included description of one or more possibleimplementations and certain variations and modifications, othervariations and modifications are within the scope of the invention,e.g., as may be within the skill and knowledge of those in the art afterunderstanding the present disclosure. It is intended to obtain rightswhich include alternative embodiments to the extent permitted, includingalternate, interchangeable and/or equivalent structures, functions,ranges or steps to those claimed, whether or not such alternate,interchangeable and/or equivalent structures, functions, ranges or stepsare disclosed herein, and without intending to publicly dedicate anypatentable subject matter. Furthermore, any feature described or claimedwith respect to any disclosed implementation may be combined in anycombination with one or more of any other features of any otherimplementation or implementations, to the extent that the features arenot necessarily technically compatible, and all such combinations arewithin the scope of the present invention.

The terms “comprising”, “containing”, “including” and “having”, andgrammatical variations of those terms, are intended to be inclusive andnonlimiting in that the use of such terms indicates the presence of somecondition or feature, but not to the exclusion of the presence also ofany other condition or feature. The use of the terms “comprising”,“containing”, “including” and “having”, and grammatical variations ofthose terms in referring to the presence of one or more components,subcomponents or materials, also include and is intended to disclose themore specific embodiments in which the term “comprising”, “containing”,“including” or “having” (or the variation of such term) as the case maybe, is replaced by any of the narrower terms “consisting essentially of”or “consisting of” or “consisting of only” (or the appropriategrammatical variation of such narrower terms). For example, the astatement that some thing “comprises” a stated element or elements isalso intended to include and disclose the more specific narrowerembodiments of the thing “consisting essentially of” the stated elementor elements, and the thing “consisting of” the stated element orelements. Examples of various features have been provided for purposesof illustration, and the terms “example”, “for example” and the likeindicate illustrative examples that are not limiting and are not to beconstrued or interpreted as limiting a feature or features to anyparticular example. The term “at least” followed by a number (e.g., “atleast one”) means that number or more than that number. The term at “atleast a portion” means all or a portion that is less than all. The term“at least a part” means all or a part that is less than all.

What is claimed is:
 1. An apparatus for collection of human biological material and post-collection processing of collected material, the apparatus being orientable in an access orientation, as oriented in the access orientation the apparatus comprising: a filter; a container having an internal containment volume having a volume in a range of from 100 cubic centimeters to 1300 cubic centimeters, the internal containment volume comprising: a tissue retention volume and a filtrate volume separated by the filter; a collection volume within the filtrate volume, the collection volume having a bottom elevation corresponding to a bottom elevation of the filtrate volume and a top elevation that is lower than the bottom elevation of the tissue retention volume; a tapered portion that tapers in a downward direction with at least a portion of the tapered portion being located below the bottom elevation of the tissue retention volume; an inlet port in fluid communication with the tissue retention volume and configured for introducing human biological material directly into the tissue retention volume; a suction port in fluid communication with the filtrate volume and providing access to the filtrate volume for suctioning from the filtrate volume components passing through the filter from the tissue retention volume to the filtrate volume; and an extraction port in fluid communication with the internal containment volume and configured for removing processed biological material from the internal containment volume; wherein each of the inlet port, the suction port and the extraction port is configured for access therethrough from above the container into the internal containment volume; a mixing device disposed at least in part within the tissue retention volume for mixing contents within the tissue retention volume, wherein the mixing device comprises a rotatable shaft extending from outside of the internal containment volume to inside of the internal containment volume, wherein the shaft is rotatable about an axis that extends through the collection volume, the apparatus further comprising: a lumen extending through the rotatable shaft and having a proximal end located outside of the internal containment volume and a distal end located within the internal containment volume, thereby providing access from outside of the internal containment volume to inside of the internal containment volume.
 2. An apparatus according to claim 1, wherein the shaft extends through the extraction port and apparatus is configured for advancing a hypodermic needle through the lumen through the extraction port to access the collection volume with an advancing tip of the hypodermic needle.
 3. An apparatus according to claim 2, wherein a distal end of the lumen is located above a portion of the filter, so that the advancing tip of a hypodermic needle pierces the filter when the tip of a hypodermic needle is advanced from the distal end of the lumen extraction port into the collection volume.
 4. An apparatus according to claim 2, wherein the collection volume includes a nadir and the distal end of the lumen is positioned above the nadir so that the tip of a hypodermic needle inserted through the lumen may be advanced vertically downward to the vicinity of the nadir of the collection volume.
 5. An apparatus according to claim 1, wherein the suction port is in fluid communication with the tapered portion of the internal containment volume through a conduit providing fluid communication from the suction port to a location within the filtrate volume and within the tapered portion of the internal containment volume.
 6. An apparatus according to claim 5, wherein: the suction port is a first suction port, the conduit is a first conduit and the location within the filtrate volume is a first location within the filtrate volume; the apparatus comprises a second suction port through which components passing through the filter from the tissue retention volume to the filtrate volume may be suctioned from the filtrate volume; and the apparatus comprises a second conduit extending from the second suction port to a second location within the filtrate volume, wherein the second conduit is translatable through the second suction port to adjust an elevation of the second location within the filtrate volume.
 7. An apparatus according to claim 1, wherein all access to the internal containment volume is through access ports wherein each said access port is configured for access through the said access port from above the container.
 8. An apparatus according to claim 7, wherein each said access port is configured for access through the said access port in a vertical direction from above the container.
 9. An apparatus according to claim 1, wherein the container comprises: a fluid containment shell with an internal cavity portion forming at least a part of the internal containment volume, the internal cavity portion being open to above; and a lid attached to the shell and disposed to cover from above the internal cavity portion; wherein, the suction port passes through the lid.
 10. An apparatus according to claim 9, wherein all access into the internal containment volume is through one or more openings passing through the lid.
 11. An apparatus according to claim 9, wherein the filter is suspended from the lid into the tissue retention volume.
 12. An apparatus according to claim 9, wherein: the shell comprises walls around the internal cavity portion except where the cavity portion is open to above; and the apparatus is configured with no access into the internal containment volume through the walls of the shell.
 13. An apparatus according to claim 9, wherein: the shell comprises an upper portion having a first wall surface portion defining a corresponding upper portion of the internal containment volume, wherein substantially all of the first wall surface portion has an incline relative to horizontal of at least 75°; and the shell comprises a lower portion located below the upper portion and having a second wall surface portion defining a corresponding lower portion of the internal containment volume, the lower portion comprising a tapered wall surface portion defining the tapered portion of the internal containment volume, substantially all of the tapered wall surface portion having an incline relative to horizontal in a range of from 30° to 60°.
 14. An apparatus according to claim 13, wherein the tapered portion of the internal containment volume occupies substantially the entire lower portion of the internal containment volume.
 15. An apparatus according to claim 1, wherein the tapered portion of the internal containment volume has a tapered portion nadir corresponding with a bottom elevation of the internal containment volume.
 16. An apparatus according to claim 1, wherein the internal containment volume has an available processing volume for receiving and processing fluids and the available processing volume is at least 100 cubic centimeters and is no larger than 1000 cubic centimeters.
 17. An apparatus according to claim 16, wherein the collection volume comprises no more than 5 percent of the available processing volume.
 18. An apparatus according to claim 17, wherein the tissue retention volume comprises at least 70 percent of the available processing volume.
 19. An apparatus according to claim 1, wherein the collection volume is no larger than 30 cubic centimeters.
 20. An apparatus according to claim 1, wherein the apparatus is containable within a first envelope volume defined by a rectangular cuboid having a length dimension of no more than 16 centimeters, a depth dimension of no more than 15 centimeters and a height dimension of no more than 18 centimeters.
 21. An apparatus according to claim 20, wherein the apparatus is not containable within a second envelope volume defined by a rectangular cuboid having any one of a length dimension, depth dimension or height dimension that is smaller than 10 centimeters.
 22. An apparatus according to claim 1, wherein the filter has a separation size in a range of from 70 microns to 800 microns.
 23. An apparatus according to claim 1, wherein the apparatus is configured to be received by a centrifuge for centrifuging.
 24. An apparatus according to claim 1, comprising human biological material disposed within the internal containment volume.
 25. An apparatus according to claim 1, comprising, disposed within the in the collection volume, a stromal vascular fraction from adipose tissue.
 26. An apparatus according to claim 1, wherein the shaft comprises a handle interface outside of the internal containment volume; and the apparatus comprises a handle interfaced to the handle interface, wherein rotating the handle causes the shaft to rotate.
 27. An apparatus according to claim 1, further comprising a removable plug disposed in a proximal end of the lumen, wherein the removable plug seals the lumen.
 28. An apparatus according to claim 1, wherein the lumen provides access to the collection volume for aspiration therefrom and injection thereto.
 29. An apparatus according to claim 1, wherein the mixing device comprises at least one mixing member disposed in the tissue retention volume and connected with the shaft, wherein the at least one mixing member moves through the tissue retention volume when the shaft is rotated.
 30. An apparatus according to claim 29, wherein at least a portion of the tissue retention volume is within the tapered portion of the internal containment volume and at least a portion of the at least one mixing member is disposed within the tapered portion of the internal containment volume.
 31. An apparatus according to claim 30, wherein when the mixing device comprises a filter contact member that moves when the shaft is rotated, the filter contact member contacting the filter at least periodically when the shaft is rotated.
 32. An apparatus according to claim 31, wherein when the shaft is rotated, the filter contact member continuously contacts the filter.
 33. An apparatus according to claim 1, wherein the apparatus is configured for advancing a hypodermic needle through the lumen and out of the distal end of the lumen to access the collection volume with an advancing tip of the hypodermic needle, wherein the distal end of the lumen is located in the tissue retention volume above a portion of the filter so that the advancing tip of the hypodermic needle pierces the filter when the tip of the hypodermic needle exits the distal end of the lumen and is advanced from the distal end of the lumen into the collection volume, and wherein the collection volume includes a nadir and an axis of the lumen is aligned so that the tip of a hypodermic needle exiting the distal end of the lumen may be advanced to the vicinity of the nadir of the collection volume. 